Target display on minicells

ABSTRACT

The invention provides compositions and methods for the production of achromosomal and anucleate cells useful for applications such as diagnositic and therapeutic uses, as well as research tools and agents for drug discovery.

RELATED APPLICATIONS

[0001] This application claims priority to the following U.S. patentapplications:

[0002] Serial No. 60/295,566 entitled “Minicell Compositions andMethods” by Roger Sabbadini, filed May 24, 2001;

[0003] Serial No. 60/359,843 entitled “Minicell Compositions andMethods” by Sabbadini, et al., filed Feb. 25, 2002; and

[0004] Serial No. ______ (attorney docket No. 089608-0401), entitled“Methods of Making Minicells,” by Surber, et al., filed May 24, 2002.

[0005] All of the preceding applications are hereby incorporated intheir entirety (including drawings) by reference thereto.

FIELD OF THE INVENTION

[0006] The invention is drawn to compositions and methods for theproduction of achromosomal archeabacterial, eubacterial and anucleateeukaryotic cells that are used as, e.g., therapeutics and/ordiagnostics, reagents in drug discovery and functional proteomics,research tools, and in other applications as well.

BACKGROUND OF THE INVENTION

[0007] The following description of the background of the invention isprovided to aid in understanding the invention, but is not admitted todescribe or constitute prior art to the invention. The contents of thearticles, patents, and patent applications, and all other documents andelectronically available information mentioned or cited in thisapplication, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other documents.

[0008] Minicells are achromosomal cells that are products of aberrantcell division, and contain RNA and protein, but little or no chromosomalDNA. Clark-Curtiss and Curtiss III, Analysis of Recombinant DNA UsingEscherichia coli Minicells, 101 Methods in Enzymology 347 (1983); Reeveand Mendelson, Minicells of Bacillus subtilis. A new system fortransport studies in absence of macromolecular biosynthesis, 352Biochim. Biophys. Acta 298-305 (1974). Minicells are capable ofplasmid-directed synthesis of discrete polypeptides in the absence ofsynthesis directed by mRNA from the bacterial chromosome. Meagher etal., Protein Expression in E. coli Minicells by Recombinant Plasmids, 10Cell 521, 523 (1977); Roozen et al., Synthesis of Ribonucleic Acid andProtein in Plasmid-Containing Minicells of Escherichia coli K-12, 107(1)J. of Bacteriology 21 (1971); and Curtiss III, Research on bacterialconjugation with minicells and minicell-producing E. coli strains, In:Microbial Drug Resistance, Editors Susumu Mitsuhashi & Hajime Hashimoto,p. 169 (Baltimore: University Park Press 1976). Early descriptions ofminicells include those of Adler et al., Genetic control of celldivision in bacteria, 154 Science 417 (1966), and Adler et al.(Miniature Escherichia coli cells deficient in DNA, 57 Proc. Nat. Acad.Sci (Wash.) 321 (1967)). However, discovery of the production ofminicells can arguably be traced to the 1930's (Frazer and Curtiss III,Production, Properties and Utility of Bacterial Minicells, 69 Curr. Top.Microbiol. Immnunol. 1-3 (1975)).

[0009] Prokaryotic (a.k.a. eubacterial) minicells have been used toproduce various eubacterial proteins. See, e.g., Michael Gaâel, et al.,The kdpF Subunit is Part of the K+-translocating Kdp Complex ofEscherichia coli and Is Responsible for Stabilization of the Complex invitro, 274(53) Jn. of Biological Chemistry 37901 (1999); Harlow, et al.,Cloning and Characterization of the gsk Gene Encoding Guanosine Kinaseof Escherichia coli, 177(8) J. of Bacteriology 2236 (1995); Carol L.Pickett, et al., Cloning, Sequencing, and Expression of the Escherichiacoli Cytolethal Distinding Toxin Genes, 62(3) Infection & Immunity 1046(1994); Raimund Eck & Jörn Belter, Cloning and characterization of agene coding for the catechol 1,2 dioxygenase of Arthrobacter sp. mA3,123 Gene 87 (1993); Andreas Schlössser, et al, Subcloning, NucleotideSequence, and Expression of trkG, a Gene That Encodes an IntegralMembrane Protein Involved in Potassium Uptake via the Trk System ofEscherichia coli, 173(10) J. of Bacteriology 3170 (1991); MehrdadJannatipour, et al., Translocation of Vibrio harveyiN,N′-Diacetylchitobiase to the Outer Membrane of Escherichia coli 169(8)J. of Bacteriology 3785 (1987); and Jacobs et al., Expression ofMycobacterium leprae genes from a Streptococcus mutans promoter inEscherichia coli K-12, 83(6) Proc. Natl. Acad. Sci. USA 1926 (1986);

[0010] Various bacteria have been used, or proposed to be used, as genedelivery vectors to mammalian cells. For reviews, see Grillot-Courvalinet al., Bacteria as gene delivery vectors for mammalian cells, 10Current Opinion in Biotechnology 477 (1999); Johnsen et al., Transfer ofDNA from Genetically Modified Organisms (GMOs), BiotechnologicalInstitute, 1-70 (2000); Sizemore et al., Attenuated Shigella as a DNAdelivery vehicle for DNA-mediated immunization, 270(5234) Science 299(1995); Patrice Courvalin, et al., Gene transfer from bacteria tomammalian cells, 318 C. R. Acad. Sci. 1207 (1995); Sizemore, et al.Attenuated bacteria as a DNA delivery vehicle for DNA-mediatedimmunization, 15(8) Vaccine 804 (1997).

[0011] U.S. Pat. No. 4,190,495, which issued Feb. 26, 1980, to Curtissis drawn to minicell producing strains of E. coli that are stated to beuseful for the recombinant expression of proteins.

[0012] U.S. Pat. No. 4,311,797, which issued Jan. 19, 1982 toKhachatourians is stated to be drawn to a minicell based vaccine. Thevaccine is stated to induce the production of antibodies againstenteropathogenic E. coli cells in cattle and is stated to be effectiveagainst coli form enteritis.

[0013] Eubacterial minicells expressing immunogens from otherprokaryotes have been described. Purcell et al., Molecular cloning andcharacterization of the 15-kilodalton major immunogen of Treponemapallidum, Infect. Immun. 57:3708, 1989.

[0014] In “Biotechnology: Promise . . . and Peril” (IDRC Reports 9:4-7,1980) authors Fleury and Shirkie aver that George Khachatourians at theUnveristy of Saskatchewan, Canada, “is working on a vaccine againstcholera using ‘minicells.’” The minciells are said to contain “genesfrom the pathogenic agent,” and the “pathogen antigens are carried onthe surface of the minicells” (p. 5, paragraph brigding the central andright columns).

[0015] Lundstrom et al., Secretion of Semliki Forest virus membraneglycoprotein E1 from Bacillus subtilis, Virus Res. 2:69-83, 1985,describe the expression of the E1 protein of the eukaryotic virus,Semliki Forest virus (SFV), in Bacillus minicells. The SFV E1 proteinused in these studies is not the native E1 protein. Rather, it is afusion protein in which the N-terminal.signal sequence and C-terminaltransmembrane domain have been removed and replaced with signalsequences from a gene from Bacillus anyloliquefaciens. The authors averthat “E1 is properly translocated through the cell membrane andsecreted” (p. 81, 1.1. 19-20), and note that “it has been difficult toexpress viral membrane proteins in prokaryotes” (p. 81, 1. 27).

[0016] U.S. Pat. No. 4,237,224, which issued Dec. 2, 1980, to Cohen andBoyer, describes the expression of X. Laevis DNA in E. coli minicells.

[0017] U.S. patent application Serial No. 60/293,566 (attorney docketNos. 078853-0401 and 089608-0201), is entitled “Minicell Compositionsand Methods,” and was filed May 24, 2001, by Sabbadini, Roger A.,Berkley, Neil L., and Klepper, Robert E., and is hereby incorporated inits entirety by reference.

[0018] Jespersen et al. describes the use of “proteoliposomes” togenerate antibodies to the AMPA receptor. Jespersen L K, Kuusinen A,Orellana A, Keinanen K, Engberg J. Use of proteoliposomes to generatephage antibodies against native AMPA receptor. Eur J Biochem. March2000, 267(5):1382-9

SUMMARY OF THE INVENTION

[0019] The invention is drawn to compositions and methods for theproduction and use of minicells, including but not limited toeubacterial minicells, in applications such as diagnostics,therapeutics, research, compound screening and drug discovery, as wellas agents for the delivery of nucleic acids and other bioactivecompounds to cells.

[0020] Minicells are derivatives of cells that lack chromosomal DNA andwhich are sometimes referred to as anucleate cells. Because eubacterialand archeabacterial cells, unlike eukaryotic cells, do not have anucleus (a distinct organelle that contains chromosomes), thesenon-eukaryotic minicells are more accurately described as being “withoutchromosomes” or “achromosomal,” as opposed to “anucleate.” Nonetheless,those skilled in the art often use the term “anucleate” when referringto bacterial minicells in addition to other minicells. Accordingly, inthe present disclosure, the term “minicells” encompasses derivatives ofeubacterial cells that lack a chromosome; derivatives of archeabacterialcells that lack their chromosome(s), and anucleate derivatives ofeukaryotic cells. It is understood, however, that some of the relevantart may use the terms “anucleate minicells” or anucleate cells” looselyto refer to any of the preceeding types of minicells.

[0021] In one aspect, the invention is drawn to a eubacterial minicellcomprising a membrane protein that is not naturally found in aprokaryote, i.e., a membrane protein from a eukaryote or anarcheabacterium. Such minicells may, but need not, comprise anexpression element that encodes and expresses the membrane protein thatit comprises. The membrane protein may be one found in anynon-eubacterial membrane, including, by way of non-limiting example, acellular membrane, a nuclear membrane, a nucleolar membrane, a membraneof the endoplasmic reticulum (ER), a membrane of a Golgi body, amembrane of a lysosome a membrane of a peroxisome, a caveolar membrane,an outer membrane of a mitochondrion or a chloroplast, and an innermembrane of a mitochondrion or a chloroplast. By way of non-limitingexample, a membrane protein may be a receptor, such as a G-proteincoupled receptor; an enzyme, such as ATPase or adenylate cyclase, acytochrome; a channel; a transporter; or a membrane-bound nucleic acidbinding factor, such as a transcription and/or translation factor;signaling components; components of the electon transport chain (ETC);or cellular antigens. A membrane fusion protein, which is generated invitro using molecular cloning techniques, does not occur in nature andis thus a membrane protein that is not naturally found in a prokaryote,even if the fusion protein is prepared using amino acid sequencesderived from eubacterial proteins.

[0022] Minicells that have segregated from parent cells lack chromosomaland/or nuclear components, but retain the cytoplasm and its contents,including the cellular machinery required for protein expression.Although chromosomes do not segregate into minicells, extrachromosomaland/or episomal genetic expression elements will segregate, or may beintroduced into mincells after segregation from parent cells. Thus, inone aspect, the invention is drawn to minicells comprising an expressionelement, which may be an inducible expression element, that comprisesexpression sequences operably linked to an open reading frame (ORF) thatencodes the non-eubacterial membrane protein. In a related aspect, theinvention is drawn to minicell-producing host cells having an expressionelement, which may be an inducible expression element, that comprisesexpression sequences operably linked to an ORF that encodes anon-eubacterial membrane protein. In a related aspect, the invention isdrawn to a method of making a eubacterial minicell comprising a membraneprotein that is not naturally found in a prokaryote, the methodcomprising growing minicell-producing host cells, the host cells havingan expression element, which may be an inducible expression element,that comprises expression sequences operably linked to an ORF thatencodes a non-eubacterial membrane protein; and preparing minicells fromthe host cells. Optionally, at any point in the method, an inducingagent is provided in order to induce expression of an ORF that encodes anon-eubacterial membrane protein.

[0023] In one aspect, the invention is drawn to display producedmembrane-associated protein(s) on the surface of the minicell. Forpurposes of this document, the term “display” is defined as exposure ofthe structure of interest on the outer surface of the minicell. By wayof non-limiting example, this structure may be an internally expressedmembrane protein or chimeric construct to be inserted in or associatedwith the minicell membrane such that the extracellular domain or domainof interest is exposed on the outer surface of the minicell (expressedand displayed on the surface of the minicell or expressed in theparental cell to be displayed on the surface of the segregatedminicell). In any scenario, the “displayed” protein or protein domain isavailable for interaction with extracellular components. Amembrane-associated protein may have more than one extracellular domain,and a minicell of the invention may display more than onemembrane-associated protein.

[0024] A membrane protein displayed by eubacterial minicells may be areceptor. Receptors include, by way of non-limiting example, G-coupledprotein receptors, hormone receptors, and growth factor receptors.Minicells displaying a receptor may, but need not, bind ligands of thereceptor. In therapeutic applications of -this aspect of the invention,the ligand is an undesirable compound that is bound to its receptor and,in some aspects, is internalized or inactivated by the minicells. Indrug discovery applications of this aspect of the invention, the ligandfor the receptor may be detectably labeled so that its binding to itsreceptor may be quantified. In the latter circumstance, the minicellsmay be used to identify and isolate, from a pool of compounds, one ormore compounds that inhibit or stimulate the activity of the receptor.That is, these minicells can be used in screening assays, includingassays such as those used in high throughput screening (HTS) systems andother drug discovery methods, for the purpose of preparing compoundsthat influence the activity of a receptor of interest.

[0025] The displayed domain of a membrane protein may be an enzymaticdomain such as on having oxidoreductase, transferase, hydrolase, lyase,isomerase ligase, lipase, kinase, phosphatase, protease, nuclease and/orsynthetase activity. Contacting such minicells with the appropriatesubstrate of the enzyme allows the substrate to be converted toreactant. When either the substrate or reactant is detectable, thereaction catalyzed by the membrane-bound enzyme may be quantified. Inthe latter instance, the minicells may be used to identify and isolate,from a pool of compounds, one or more compounds that inhibit orstimulate the activity of the enzyme represented by the displayedenzymatic moiety. That is, these minicells can be used in screeningassays, including assays such as those used in high throughput screening(HTS) systems and other drug discovery methods, for the purpose ofpreparing compounds that influence the activity of an enzyme orenzymatic moiety of interest.

[0026] The membrane protein displayed by minicells may be a fusionprotein, i.e., a protein that comprises a first polypeptide having afirst amino acid sequence and a second polypeptide having a second aminoacid sequence, wherein the first and second amino acid sequences are notnaturally present in the same polypeptide. At least one polypeptide in amembrane fusion protein is a “transmembrane domain” or“membrane-anchoring domain”. The transmembrane and membrane-anchoringdomains of a membrane fusion protein may be selected from membraneproteins that naturally occur in a eucaryote, such as a fungus, aunicellular eucaryote, a plant and an animal, such as a mammal includinga human. Such domains may be from a viral membrane protein naturallyfound in a virus such as a bacteriophage or a eucaryotic virus, e.g., anadenovirus or a retrovirus. Such domains may be from a membrane proteinnaturally found in an archaebacterium such as a thermophile.

[0027] The displayed domain of a membrane fusion protein may be anenzymatic domain such as one having oxidoreductase, transferase,hydrolase, lyase, isomerase ligase, lipase, kinase, phosphatase,protease, nuclease and/or synthetase activity. Contacting such minicellswith the appropriate substrate of the enzyme allows the substrate to beconverted to reactant. When either the substrate or reactant isdetectable, the reaction catalyzed by the membrane-bound enzyme may bequantified. In the latter instance, the minicells may be used toidentify and isolate, from a pool of compounds, one or more compoundsthat inhibit or stimulate the activity of the enzyme represented by thedisplayed enzymatic moiety. That is, these minicells can be used inscreening assays, including assays such as those used in high throughputscreening (HTS) systems and other drug discovery methods, for thepurpose of preparing compounds that influence the activity of an enzymeor enzymatic moiety of interest.

[0028] The displayed domain of a membrane fusion protein may be abinding moiety. By way of non-limiting example, binding moieties usedfor particular purposes may be a binding moiety directed to a compoundor moiety displayed by a specific cell type or cells found predominantlyin one type of tissue, which may be used to target minicells and theircontents to specific cell types or tissues; or a binding moiety that isdirected to a compound or moiety displayed by a pathogen, which may beused in diagnostic or therapeutic methods; a binding moiety that isdirected to an undesirable compound, such as a toxin, which may be usedto bind and preferably internalize and/or neutralize the undesirablecompound; a diseased cell; or the binding moiety may be a domain thatallows for the minicells to be covalently or non-covalently attached toa support material, which may be used in compositions and methods forcompound screening and drug discovery. By “diseased cell” it is meantpathogen-infected cells, malfunctioning cells, and dysfunctional cells,e.g., cancer cells.

[0029] In various aspects, the minicells of the invention comprise oneor more biologically active compounds. The term “biologically active”(synonymous with “bioactive”) indicates that a composition or compounditself has a biological effect, or that it modifies, causes, promotes,enhances, blocks, reduces, limits the production or activity of, orreacts with or binds to an endogenous molecule that has a biologicaleffect. A “biological effect” may be but is not limited to one thatstimulates or causes an immunoreactive response; one that impacts abiological process in an animal; one that impacts a biological processin a pathogen or parasite; one that generates or causes to be generateda detectable signal; and the like. Biologically active compositions,complexes or compounds may be used in therapeutic, prophylactic anddiagnostic methods and compositions. Biologically active compositions,complexes or compounds act to cause or stimulate a desired effect uponan animal. Non-limiting examples of desired effects include, forexample, preventing, treating or curing a disease or condition in ananimal suffering therefrom; limiting the growth of or killing a pathogenin an animal infected thereby; augmenting the phenotype or genotype ofan animal; stimulating a prophylactic immnunoreactive response in ananimal; or diagnosing a disease or disorder in an animal.

[0030] In the context of therapeutic applications of the invention, theterm “biologically active” indicates that the composition, complex orcompound has an activity that impacts an animal suffering from a diseaseor disorder in a positive sense and/or impacts a pathogen or parasite ina negative sense. Thus, a biologically active composition, complex orcompound may cause or promote a biological or biochemical activitywithin an animal that is detrimental to the growth and/or maintenance ofa pathogen or parasite; or of cells, tissues or organs of an animal thathave abnormal growth or biochemical characteristics, such as cancercells.

[0031] In the context of diagnostic applications of the invention, theterm “biologically active” indicates that the composition, complex orcompound can be used for in vivo or ex vivo diagnostic methods and indiagnostic compositions and kits. For diagnostic purposes, a preferredbiologically active composition or compound is one that can be detected,typically (but not necessarily) by virtue of comprising a detectablepolypeptide. Antibodies to an epitope found on composition or compoundmay also be used for its detection.

[0032] In the context of prophylactic applications of the invention, theterm “biologically active” indicates that the composition or compoundinduces or stimluates an immunoreactive response. In some preferredembodiments, the immunoreactive response is designed to be prophylactic,i.e., prevents infection by a pathogen. In other preferred embodiments,the immunoreactive response is designed to cause the immune system of ananimal to react to the detriment of cells of an animal, such as cancercells, that have abnormal growth or biochemical characteristics. In thisapplication of the invention, compositions, complexes or compoundscomprising antigens are formulated as a vaccine.

[0033] It will be understood by those skilled in the art that a givencomposition, complex or compound may be biologically active intherapeutic, diagnostic and prophylactic applications. A composition,complex or compound that is described as being “biologically active in acell” is one that has biological activity in vitro (i.e., in a cellculture) or in vivo (i.e., in the cells of an animal). A “biologicallyactive component” of a composition or compound is a portion thereof thatis biologically active once it is liberated from the composition orcompound. It should be noted, however, that such a component may also bebiologically active in the context of the composition or compound.

[0034] In one aspect, the minicells of the invention comprise atherapeutic agent. Such minicells may be used to deliver therapeuticagents. In a preferred embodiment, a minicell comprising a therapeuticagent displays a binding moiety that specifically binds a ligand presenton the surface of a cell, so that the minicells may be “targeted” to thecell. The therapeutic agent may be any type of compound or moiety,including without limitation small molecules, polypeptides, antibodiesand antibody derivatives and nucleic acids. The therapeutic agent may bea drug; a prodrug, i.e., a compound that becomes biologically active invivo after being introduced into a subject in need of treatment; or animmunogen.

[0035] In one aspect, the minicells of the invention comprise adetectable compound or moiety. As is understood by those of skill in theart, a compound or moiety that is “detectable” produces a signal thatcan detected by spectroscopic, photochemical, biochemical,immunochemical, electromagnetic, radiochemical, or chemical means suchas fluorescence, chemifluoresence, or chemiluminescence,electrochemilumenscence, or any other appropriate means. A detectablecompound may be a detectable polypeptide, and such polypeptides may, butneed not, be incorporated into fusion membrane proteins of the minicell.Detectable polypeptides or amino acid sequences, includes, by way ofnon-limiting example, a green fluorescent protein (GFP), a luciferase, abeta-galactosidase, a His tag, an epitope, or a biotin-binding proteinsuch as streptavidin or avidin. The detectable compound or moiety may bea radiolabeled compound or a radioisotope. A detectable compound ormoiety may be a small molecule such as, by way of non-limiting example,a fluorescent dye; a radioactive iostope; or a compound that may bedetected by x-rays or electromagnetic radiation. Image enhancers asthose used for CAT and PET scans (e.g., calcium, gallidium) may be used.In another non-limiting example, detectable labels may also include lossof catalytic substrate or gain of catalytic product following catalysisby a minicell displayed, solule cytoplasmic, or secreted enzyme.

[0036] In one aspect, the invention is drawn to a minicell comprisingone or more bioactive nucleic acids or templates thereof. By way ofnon-limiting example, a bioactive nucleic acid may be an antisenseoligonucleotide, an aptamer, an antisense transcript, a ribosomal RNA(rRNA), a transfer RNA (tRNA), a molecular decoy, or an enzymaticallyactive nucleic acid, such as a ribozyme. Such minicells can, but neednot, comprise a displayed polypeptide or protein on the surface of theminicell. The displayed polypeptide or protein may be a binding moietydirected to a compound or moiety displayed by a particular type of cell,or to a compound or moiety displayed by a pathogen. Such minicells canfurther, but need not, comprise an expression element havingeubacterial, archael, eucaryotic, or viral expression sequences operablylinked to a nucleotide sequence that serves as a template for abioactive nucleic acid.

[0037] In one aspect, the invention is drawn to immunogenic minicells,i.e., minicells that display an immunogen, vaccines comprisingimmunogenic minicells, antibodies and antibody derivatives directed toimmunogens displayed on immunogenic minicells, and method of making andusing immunogenic minicells and antibodies and antibody derivativesproduced therefrom in prophylactic, diagnostic, therapeutic and researchapplications. A preferred immunogen displayed by a minicell is animmunogenic polypeptide, which is preferably expressed from anexpression element contained within the minicell in order to maximizethe amount of immunogen displayed by the immunogenic minicells. Theimmunogenic polypeptide can be derived from any organism, obligateintracelluar parasite, organelle or virus with the provisio that, inprophylactic applications, the immunogenic polypeptide is not derivedfrom a prokaryote, including a eubacterial virus. The source organismfor the immunogen may be a pathogen. A minicell displaying an immunogenderived from a pathogen is formulated into a vaccine and, in aprophylactic application, used to treat or prevent diseases anddisorders caused by or related to the eukaryotic or archeabacterialpathogen.

[0038] In a separate aspect, the invention is drawn to minicells thatdisplay an immunogen derived from a nonfunctional, dysfunctional and/ordiseased cell. By way of non-limiting example, the minicells display animmunogenic polypeptide derived from a hyperproliferative cell, i.e., acell that is tumorigenic, or part of a tumor or cancer. As anothernon-limiting example, a cell that is infected with a virus or anobligate intracellular parasite (e.g., Rickettsiae) displays animmunogenic polypeptide that is encoded by the genome of the infectedcell but is aberrenatly expressed in an infected cell. A vaccinecomprising a minicell displaying an immunogen derived from anonfunctional, dysfunctional and/or diseased cell is used in methods oftreating or preventing hyperproliferative diseases or disorders,including without limitation a cell comprising an intracellularpathogen.

[0039] In one aspect, the invention is drawn to methods of usingminicells, and expression systems optimized therefore, to manufacture,on a large scale, proteins using recombinant DNA technology. In arelated aspect, the invention is drawn to the production, viarecombinant DNA technology, and/or segration of exogenous proteins inminicells. The minicells are enriched for the exogenous protein, whichis desirable for increased yield and purity of the protein. In additionto protein purification, the minicells can be used for crystallography,the study of intracellular or extracellular protein-proteininteractions, the study of intracellular or extracellularprotein-nucleic acid interactions, the study of intracellular orextracellular protein-membrane interactions, and the study of otherbiological, chemical, or physiological event(s).

[0040] In one aspect, the invention is drawn to minicells having amembrane protein that has an intracellular domain. By way ofnon-limiting example, the intracellular domain is exposed on the innersurface of the minicell membrane oriented towards the cytoplasmiccompartment. The intracellular protein domain is available forinteraction with intracellular components. Intracellular components maybe naturally present in the minicells or their parent cells, or may beintroduced into minicells after segregation from parent cells. Amembrane-associated protein may have more than one intracellular domain,and a minicell of the invention may display more than onemembrane-associated protein.

[0041] In one aspect, the invention is drawn to a minicell comprising amembrane protein that is linked to a conjugatable compound (a.k.a.“attachable compound”). The conjugatable compound may be of any chemicalnature and have one or more therapeutic or detectable moities. By way ofnon-limiting example, a protein having a transmembrane or membraneanchoring domain is displayed and has the capacity to be specificallycross-linked on its extracellular domain. Through this approach, anyconjugatable compound of interest may be quickly and easily attached tothe outer surface of minicells containing this expressedmembrane-spanning domain. In aspects of the invention wherein minicellsare used for drug delivery in vivo, a preferred conjugatable compound ispolyethylene glycol (PEG, which provides for “stealth” minicells thatare not taken as well and/or as quickly by the reticuloendothelialsystem (RES). Other conjugatable compounds include polysaccharides,polynucleotides, lipopolysaccharides, lipoproteins, glycosylatedproteins, synthetic chemical compounds, and/or chimeric combinations ofthese examples listed.

[0042] In various aspects of the invention, the minicell displays apolypeptide or other compound or moiety on its surface. By way ofnon-limiting example, a non-eubacterial membrane protein displayed byeubacterial minicells may be a receptor. Minicells displaying a receptormay, but need not, bind ligands of the receptor. In therapeuticapplications of this aspect of the invention, the ligand is anundesirable compound that is bound to its receptor and, in some aspects,is internalized by the minicells. In drug discovery applications of thisaspect of the invention, the ligand for the receptor may be detectablylabeled so that its binding to its receptor may be quantified. In thelatter circumstance, the minicells may be used to identify and isolate,from a pool of compounds, one or more compounds that inhibit orstimulate the activity of the receptor. That is, these minicells can beused in screening assays, including assays such as those used in highthroughput screening (HTS) systems and other drug discovery methods, forthe purpose of preparing compounds that influence the activity of areceptor of interest.

[0043] The non-eubacterial membrane protein displayed by minicells maybe a fusion protein, i.e., a protein that comprises a first polypeptidehaving a first amino acid sequence and a second polypeptide having asecond amino acid sequence, wherein the first and second amino acidsequences are not naturally present in the same polypeptide. At leastone polypeptide in a membrane fusion protein is a “transmembrane domain”or “membrane-anchoring domain”. The transmembrane and membrane-anchoringdomains of a membrane fusion protein may be selected from membraneproteins that naturally occur in a eukaryote, such as a fungus, aunicellular eukaryote, a plant and an animal, such as a mammal includinga human. Such domains may be from a viral membrane protein naturallyfound in a virus such as a bacteriophage or a eukaryotic virus, e.g., anadenovirus or a retrovirus. Such domains may be from a membrane proteinnaturally found in an archaebacterium such as a thermophile.

[0044] The displayed domain of a membrane fusion protein may be anenzymatic domain such as one having the activity of a lipase, a kinase,a phosphatase, a reductase, a protease, or a nuclease. Contacting suchminicells with the appropriate substrate of the enzyme allows thesubstrate to be converted to reactant. When either the substrate orreactant is detectable, the reaction catalyzed by the membrane-boundenzyme may be quantified. In the latter instance, the minicells may beused to identify and isolate, from a pool of compounds, one or morecompounds that inhibit or stimulate the activity of the enzymerepresented by the displayed enzymatic moiety. That is, these minicellscan be used in screening assays, including assays such as those used inhigh throughput screening (HTS) systems and other drug discoverymethods, for the purpose of preparing compounds that influence theactivity of an enzyme or enzymatic moiety of interest.

[0045] The displayed domain of a membrane fusion protein may be abinding moiety. By way of non-limiting example, binding moieties usedfor particular purposes may be a binding moiety directed to a compoundor moiety displayed by a specific cell type or cells found predominantlyin one type of tissue, which may be used to target minicells and theircontents to specific cell types or tissues; or a binding moiety that isdirected to a compound or moiety displayed by a pathogen, which may beused in diagnostic or therapeutic methods; a binding moiety that isdirected to an undesirable compound, such as a toxin, which may be usedto bind and preferably internalize and/or neutralize the undesirablecompound; a diseased cell; or the binding moiety may be a domain thatallows for the minicells to be covalently or non-covalently attached toa support material, which may be used in compositions and methods forcompound screening and drug discovery.

[0046] In one aspect, the invention provides compositions and methodsfor preparing a soluble and/or secreted protein where the proteinremains in the cytoplasm of the minicell or is secreted following nativesecretory pathways for endogenous screted proteins or is secreted usingchimeric fusion to secretory signaling sequences. By way of non-limitingexample, secreted or cytoplasmic soluble proteins may be produced forpurification, targeted therapeutic applications where the proteinproduced is a therapeutic agent and is produced at the desired site of,detection for screening or diagnostic purposes where the protein isproduced in response to a simulous and/or localization event, or tostimulate targeted minicell-cell fusion or interaction events where theprotein produced stimulates cell-cell fusion upon targeted stimulation.

[0047] In one aspect, the invention provides compositions and methodsfor preparing antibodies and/or antibody derivatives that recognize animmunogenic epitope present on the native form of a membrane protein,but which is not immunogenic when the membrane protein is denatured orwhen prepared as a synthetic oligopeptide. Such antibodies and antibodyderivatives are said to be “conformation sensitive.” Unlike mostantibodies and antibody derivatives prepared by using a denaturedmembrane protein or an oligopeptide derived from the membrane protein,conformation sensitive antibodies and antibody derivatives specificallybind membrane proteins in their native state (i.e., in a membrane) withhigh affinity. Conformation sensitive antibodies and antibodyderivatives are used to target compounds and compositions, including aminicell of the invention, to a cell displaying the membrane protein ofchoice. Conformation sensitive antibodies and antibody derivatives arealso used to prevent receptors from binding their natural ligands byspecifically binding to the receptor with a high affinity and therebylimiting access of the ligand to the receptor. Conformation sensitiveantibodies and antibody derivatives can be prepared that are specificfor a specific isoform or mutant of a membrane protein, which can beuseful in research and medical applications.

[0048] In one aspect, the invention provides biosensors comprisingminicells including, not limited to, the minicells of the invention. Anexemplary biosensor of the invention is a BIAcore chip, i.e., a chiponto which minicells are attached, where the minicells undergo somechange upon exposure to a preselected compound, and the change isdetected using surface plasmon resonance. A biosensor comprisingminicells can be used in methods of detecting the presence of anundesirable compound. Undesirable compounds include but are not limitedto, toxins; pollutants; explosives, such as those in landmines orillegally present; illegal narcotics; components of biological orchemical weapons. In a related aspect, the invention provides a devicecomprising a microchip operatively associated with a biosensorcomprising a minicell. The device can further comprise an actuator thatperforms a responsive function when the sensor detects a preselectedlevel of a marker.

[0049] In one aspect, the invention provides minicells that may be usedas research tools and/or kits comprising such research tools. Theminicells of the invention may be used as is, or incorporated intoresearch tools useful for scientific research regarding all amino acidcomprising compounds including, but not limited to membrane-associatedproteins, chimeric membrane fusion proteins, and soluble proteins. Suchscientific research includes, by way of non-limiting example, basicresearch, as well as pharmacological, diagnostic, and pharmacogeneticstudies. Such studies may be carried out in vivo or in vitro.

[0050] In one aspect, the invention is drawn to archaebacterialminicells. In a related aspect, the invention is drawn toarchaebacterial minicells comprising at least one exogenous protein,that is, a protein that is not normally found in the parent cell,including without limitation fusion proteins. The archaebacterialminicells of the invention optionally comprise an expression elementthat directs the production of the exogenous protein(s).

[0051] In other aspects, the invention is drawn to methods of preparingthe minicells, protoplasts, and poroplasts™ of the invention for variousapplications including but not limited to diagnostic, therapeutic,research and screening applications. In a related aspect, the inventionis drawn to pharmaceutical compositions, reagents and kits comprisingminicells.

[0052] In each aspect and embodiment of the invention, unless statedotherwise, embodiments wherein the minicell is a eubacterial minicell, aporoplast, a spheroplast or a protoplast exist.

[0053] In a first aspect, the invention provides a minicell comprising amembrane protein selected from the group consisting of a eukaryoticmembrane protein, an archeabacterial membrane protein and an organellarmembrane protein. In another embodiment, wherein the minicell comprisesa biologically active compound. By way of non-limiting example, thebiologically active compound is a radioisotope, a polypeptide, a nucleicacid or a small molecule.

[0054] In another embodiment, the minicell comprises a expressionconstruct, wherein the first expression construct comprises expressionsequences operably linked to an ORF that encodes a protein. In anotherembodiment, the ORF encodes the membrane protein. In another embodiment,the expression sequences that are operably linked to an ORF areinducible and/or repressible.

[0055] In another aspect, the minicell comprises a second expressionconstruct, wherein the second expression construct comprises expressionsequences operably linked to a gene. In another embodiment, theexpression sequences that are operably linked to a gene are inducibleand/or repressible. In a related embodiment, the gene product of thegene regulates the expression of the ORF that encodes the protein. Afactor that “regulates” the expression of a gene or a gene productdirectly or indirectly initiates, enhances, quickens, slows, terminates,limits or completely blocks expression of a gene. In differentembodiments, the gene product of the gene is a nucleic acid or apolypeptide. The polypeptide can be of any type, including but notlimited to a membrane protein, a soluble protein or a secreted protein.A membrane protein can be a membrane fusion protein comprising a firstpolypeptide, which comprises at least one transmembrane domain or atleast one membrane anchoring domain; and a second polypeptide.

[0056] In one aspect, the invention provides a minicell comprising amembrane fusion protein, the fusion protein comprising a firstpolypeptide, the first polypeptide comprising at least one transmembranedomain or at least one membrane anchoring domain; and a secondpolypeptide, wherein the second polypeptide is not derived from aeubacterial protein and is neither a His tag nor aglutathione-S-transferase polypeptide. In various embodiments, theminicell is a eubacterial minicell, a poroplast, a spheroplast or aprotoplast. In one embodiment, the minicell comprises a biologicallyactive compound.

[0057] In one aspect, the invention provides a minicell comprising amembrane conjugate, wherein the membrane conjugate comprises a membraneprotein chemically linked to a conjugated compound. In one embodiment,the conjugated compound is selected from the group consisting of anucleic acid, a polypeptide, a lipid and a small molecule.

[0058] In one aspect, the invention provides a method for makingminicells, comprising (a) culturing a minicell-producing parent cell,wherein the parent cell comprises an expression construct, wherein theexpression construct comprises a gene operably linked to expressionsequences that are inducible and/or repressible, and wherein inductionor repression of the gene causes or enhances the production ofminicells; and (b) separating the minicells from the parent cell,thereby generating a composition comprising minicells, wherein aninducer or repressor is present within the parent cells during one ormore steps and/or between two or more steps of the method. In oneembodiment, the method further comprises (c) purifying the minicellsfrom the composition.

[0059] Relevant gene products are factors involved in or modulating DNAreplication, cellular division, cellular partitioning, septation,transcription, translation, or protein folding. The minicells areseparated from parent cells by processes such as centrifugation,ultracentrifugation, density gradation, immunoaffinity,immunoprecipitation and other techniques described herein.

[0060] In one embodiment, the minicell is a poroplast, and the methodfurther comprises (d) treating the minicells with an agent, orincubating the minicells under a set of conditions, that degrades theouter membrane of the minicell. The outer membrane is degraded bytreatment with an agent selected from the group consisting of EDTA,EGTA, lactic acid, citric acid, gluconic acid, tartaric acid,polyethyleneimine, polycationic peptides, cationic leukocyte peptides,aminoglycosides, aminoglycosides, protamine, insect cecropins, reptilianmagainins, polymers of basic amino acids, polymixin B, chloroform,nitrilotriacetic acid and sodium hexametaphosphate; by exposure toconditions selected from the group consisting of osmotic shock andinsonation; and by other methods described herein.

[0061] In one embodiment, further comprising removing one or morecontaminants from the composition. Representative contaminants are LPSand peptidoglycan. In a representative embodiment, LPS is removed bycontacting the composition to an agent that binds or degrades LPS. Atleast about 50%, preferably about 65% to about 75%, more preferably 95%,most preferably 99% or >99% of LPS is removed from an initialpreparation of minicells. In a related embodiment, theminicell-producing parent cell comprises a mutation in a gene requiredfor lipopolysaccharide synthesis.

[0062] In on embodiment, the minicell is a spheroplast, and the methodfurther comprises (d) treating the minicells with an agent, orincubating the minicells under a set of conditions, that disrupts ordegrades the outer membrane; and (e) treating the minicells with anagent, or incubating the minicells under a set of conditions, thatdisrupts or degrades the cell wall. The agent that disrupts or degradesthe cell wall can be. e.g., a lysozyme, and the set of conditions thatdisrupts or degrades the cell wall can be, e.g., incubation in ahypertonic solution.

[0063] In one embodiment, the minicell is a protoplast, and the methodfurther comprises (d treating the minicells with an agent, or incubatingthe minicells under a set of conditions, that disrupt or degrade theouter membrane; (e) treating the minicells with an agent, or incubatingthe minicells under a set of conditions, that disrupts or degrades thecell wall, in order to generate a composition that comprisesprotoplasts; and (f) purifying protoplasts from the composition. In oneembodiment, the method further comprises preparing a denuded minicellfrom the minicell. In one embodiment, the method further comprisescovalently or non-covalently linking one or more components of theminicell to a conjugated moiety.

[0064] In one aspect, the invention provides a L-form minicellcomprising (a) culturing an L-form eubacterium, wherein the eubacteriumcomprises one or more of the following: (i) an expression element thatcomprises a gene operably linked to expression sequences that areinducible and/or repressible, wherein induction or repression of thegene regulates the copy number of an episomal expression construct; (ii)a mutation in an endogenous gene, wherein the mutation regulates thecopy number of an episomal expression construct; (iii) an expressionelement that comprises a gene operably linked to expression sequencesthat are inducible and/or repressible, wherein induction or repressionof the gene causes or enhances the production of minicells; and (iv) amutation in an endogenous gene, wherein the mutation causes or enhancesminicell production; (b) culturing the L-form minicell-producing parentcell in media under conditions wherein minicells are produced; and (c)separating the minicells from the parent cell, thereby generating acomposition comprising L-form minicells, wherein an inducer or repressoris present within the minicells during one or more steps and/or betweentwo or more steps of the method. In one embodiment, the method furthercomprises (d) purifying the L-form minicells from the composition.

[0065] In one aspect, the invention provides a method of producing aprotein, comprising (a) transforming a minicell-producing parent cellwith an expression element that comprises expression sequences operablylinked to a nucleic acid having an ORF that encodes the protein; (b)culturing the minicell-producing parent cell under conditions whereinminicells are produced; and (c) purifying minicells from the parentcell, (d) purifying the protein from the minicells, wherein the ORF isexpressed during step (b), between steps (b) and (c), and during step(c).

[0066] In one embodiment, the expression elements segregate into theminicells, and the ORF is expressed between steps (c) and (d). In oneembodiment, the protein is a soluble protein contained within theminicells, and the method further comprises (e) lysing the minicells.

[0067] In one embodiment, the protein is a secreted protein, and themethod further comprises (e) collecting a composition in which theminicells are suspended or with which the minicells are in contact.

[0068] In one embodiment, the expression sequences to which the ORF isoperably linked are inducible, wherein the method further comprisesadding an inducing agent between steps (a) and (b); during step (b); andbetween steps (b) and (c).

[0069] In one embodiment, the expression sequences to which the ORF isoperably linked are inducible, wherein the expression elements segregateinto the minicells, the method further comprises adding an inducingagent after step (c).

[0070] In one embodiment, the method further comprises (e) preparingporoplasts from the minicells, wherein the ORF is expressed during step(b); between steps (b) and (c); during step (c); between step (c) andstep (d) when the expression elements segregate into the minicells;and/or after step (d) when the expression elements segregate into theminicells.

[0071] In one embodiment, the method further comprises (f) purifying theprotein from the poroplasts.

[0072] In one embodiment, the method further comprises (e) preparingspheroplasts from the minicells, wherein the ORF is expressed duringstep (b), between steps (b) and (c), during step (c), between steps (c)and (d) and/or after step (d).

[0073] In one embodiment, the method further comprises (f) purifying theprotein from the spheroplasts.

[0074] In one embodiment, the method further comprises (e) preparingprotoplasts from the minicells, wherein the ORF is expressed during step(b), between steps (b) and (c), during step (c), between steps (c) and(d) and/or after step (d).

[0075] In one embodiment, the method further comprises (f) purifying theprotein from the protoplasts.

[0076] In one embodiment, the method further comprises (e) preparingmembrane preparations from the minicells, wherein the ORF is expressedduring step (b), between steps (b) and (c), during step (c), betweensteps (c) and (d) and/or after step (d).

[0077] In one embodiment, the method further comprises (f) purifying theprotein from the membrane preparations.

[0078] In one embodiment, the minicell-producing parent cell is anL-form bacterium.

[0079] In one aspect, the invention provides a method of producing aprotein, comprising (a) transforming a minicell with an expressionelement that comprises expression sequences operably linked to a nucleicacid having an ORF that encodes the protein; and (b) incubating theminicells under conditions wherein the ORF is expressed.

[0080] In one embodiment, the method further comprises (c) purifying theprotein from the minicells.

[0081] In one aspect, the invention provides a method of producing aprotein, comprising (a) transforming a minicell-producing parent cellwith an expression element that comprises expression sequences operablylinked to a nucleic acid having an ORF that encodes a fusion proteincomprising the protein and a polypeptide, wherein a protease-sensitiveamino acid sequence is positioned between the protein and thepolypeptide; (b) culturing the minicell-producing parent cell underconditions wherein minicells are produced; (c) purifying minicells fromthe parent cell, wherein the ORF is expressed during step (b); betweensteps (b) and (c); and/or after step (c) when the expression elementssegregate into the minicells; and (d) treating the minicells with aprotease that cleaves the sensitive amino acid sequence, therebyseparating the protein from the polypeptide.

[0082] In one aspect, the invention provides a poroplast, the poroplastcomprising a vesicle, bonded by a membrane, wherein the membrane is aneubacterial inner membrane, wherein the vesicle is surrounded by aeubacterial cell wall, and wherein the eubacterial inner membrane isaccessible to a compound in solution with the poroplast. In oneembodiment, the poroplast is a cellular poroplast. The compound has amolecular weight of at least 1 kD, preferably at least about 0.1 toabout 1 kD, more preferably from about 1, 10 or 25 kD to about 50 kD,and most preferably from about 75 or about 100 kD to about 150 or 300kD.

[0083] In one embodiment, the poroplast comprises an exogenous nucleicacid, which may be an expression construct. In one embodiment, theexpression construct comprises an ORF that encodes an exogenous protein,wherein the ORF is operably linked to expression sequences. In oneembodiment, the exogenous protein is a fusion protein, a soluble proteinor a secreted protein. In one embodiment, the exogenous protein is amembrane protein, and is preferably accessible to compounds in solutionwith the poroplast. In one embodiment, poroplasts are placed in ahypertonic solution, wherein 90% or more of an equivalent amount ofspheroplasts or protoplasts lyse in the solution under the sameconditions.

[0084] In one embodiment, the membrane protein is selected from thegroup consisting of a eukaryotic membrane protein, an archeabacterialmembrane protein, and an organellar membrane protein. In one embodiment,the membrane protein is a fusion protein, the fusion protein comprisinga first polypeptide, the first polypeptide comprising at least onetransmembrane domain or at least one membrane anchoring domain; and asecond polypeptide, wherein the second polypeptide is displayed by theporoplast. In one embodiment, the second polypeptide is displayed on theexternal side of the eubacterial inner membrane. The second polypeptidecan be an enzyme moiety, a binding moiety, a toxin, a cellular uptakesequence, an epitope, a detectable polypeptide, and a polypeptidecomprising a conjugatable moiety. An enzyme moiety is a polypeptidederived from, by way of non-limiting example, a cytochrome P450, anoxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, aligase or a synthetase.

[0085] In one embodiment, the poroplast comprises a membrane componentthat is chemically linked to a conjugated compound.

[0086] In one embodiment, the expression construct comprises one or moreDNA fragments from a genome or cDNA. In one embodiment, the exogenousprotein has a primary amino acid sequence predicted from a nucleic acidsequence.

[0087] In one aspect, the invention provides a solid support comprisinga minicell. In various embodiments, the solid support is a dipstick, abead or a mictrotiter multiwell plate. In one embodiment, the minicellcomprises a detectable compound, which may be a colorimetric,fluorescent or radioactive compound.

[0088] In one embodiment, the minicell displays a membrane componentselected from the group consisting of (i) a eukaryotic membrane protein,(ii) an archeabacterial membrane protein, (iii) an organellar membraneprotein, (iv) a fusion protein comprising at least one transmembranedomain or at least one membrane anchoring domain, and (v) a membraneconjugate comprising a membrane component chemically linked to aconjugated compound.

[0089] In one embodiment, the membrane component is a receptor. In arelated embodiment, the solid support further comprises a co-receptor.In one embodiment, the minicell displays a binding moiety.

[0090] In one aspect, the invention provides a solid support comprisinga minicell, wherein the minicell displays a fusion protein, the fusionprotein comprising a first polypeptide that comprises at least onetransmembrane domain or at least one membrane anchoring domain, and asecond polypeptide. In various embodiments, the second polypeptidecomprises a binding moiety or an enzyme moiety.

[0091] In one aspect, the invention provides a solid support comprisinga minicell, wherein the minicell comprises a membrane conjugatecomprising a membrane component chemically linked to a conjugatedcompound. In one embodiment, the conjugated compound is a spacer. In oneembodiment, the spacer is covalently linked to the solid support. In oneembodiment, the conjugated compound is covalently linked to the solidsupport.

[0092] In one aspect, the invention provides a minicell comprising abiologically active compound, wherein the minicell displays a ligand orbinding moiety, wherein the ligand or binding moiety is part of a fusionprotein comprising a first polypeptide that comprises at least onetransmembrane domain or at least one membrane anchoring domain and asecond polypeptide that comprises a binding moiety, and the minicell isa poroplast, spheroplast or protoplast.

[0093] In one aspect, the invention provides a eubacterial minicellcomprising a biologically active compound, wherein the minicell displaysa binding moiety, wherein the binding moiety is selected from the groupconsisting of (a) a eukaryotic membrane protein; (b) an archeabacterialmembrane protein; (c) an organellar membrane protein; and (d) a fusionprotein, the fusion protein comprising a first polypeptide, the firstpolypeptide comprising at least one transmembrane domain or at least onemembrane anchoring domain; and a second polypeptide, wherein the secondpolypeptide is not derived from a eubacterial protein and is neither aHis tag nor a glutathione-S-transferase polypeptide, and wherein thepolypeptide comprises a binding moiety.

[0094] In one embodiment, the binding moiety is selected from the groupconsisting of an antibody, an antibody derivative, a receptor and anactive site of a non-catalytic derivative of an enzyme. In a preferredembodiment, the binding moiety is a single-chain antibody. In oneembodiment, one of the ORFs encodes a protein that comprises the bindingmoiety.

[0095] In one embodiment, the binding moiety is directed to a ligandselected from the group consisting of an epitope displayed on apathogen, an epitope displayed on an infected cell and an epitopedisplayed on a hyperproliferative cell.

[0096] In one embodiment, the invention further comprises a first andsecond nucleic acid, wherein the first nucleic acid comprises eukaryoticexpression sequences operably linked to a first ORF, and a secondnucleic acid, wherein the second nucleic acid comprises eubacterialexpression sequences operably linked to a second ORF.

[0097] In one embodiment, the eubacterial expression sequences areinduced and/or derepressed when the binding moiety is in contact with atarget cell. In a variant embodiment, the eukaryotic expressionsequences are induced and/or derepressed when the nucleic acid is in thecytoplasm of a eukaryotic cell. In related embodiments, the proteinencoded by the first ORF comprises eukaryotic secretion sequences and/orthe protein encoded by the second ORF comprises eubacterial secretionsequences.

[0098] In one aspect, the invention provides a method of associating aradioactive compound with a cell, wherein the cell displays a ligandspecifically recognized by a binding moiety, comprising contacting thecell with a minicell that comprises the radioactive compound anddisplays the binding moiety. In a diagnostic embodiment, the amount ofradiation emitted by the radioactive isotope is sufficient to bedetectable. In a therapeutic embodiment, the amount of radiation emittedby the radioactive isotope is sufficient to be cytotoxic. In oneembodiment, the ligand displayed by the cell is selected from the groupconsisting of an epitope displayed on a pathogen, an epitope displayedon an infected cell and an epitope displayed on a hyperproliferativecell. In one embodiment, the binding moiety is selected from the groupconsisting of an antibody, an antibody derivative, a channel protein anda receptor, and is preferably a single-chain antibody. In otherembodiments, the binding moiety is an aptamer or a small molecule. Inone embodiment, the ligand is selected from the group consisting of acytokine, hormone, and a small molecule.

[0099] In one aspect, the invention provides a method of delivering abiologically active compound to a cell, wherein the cell displays aligand specifically recognized by a binding moiety, comprisingcontacting the cell with a minicell that displays the binding moiety,wherein the minicell comprises the biologically active compound, andwherein the contents of the minicell are delivered into the cell from aminicell bound to the cell. In one embodiment, the biologically activecompound is selected from the group consisting of a nucleic acid, alipid, a polypeptide, a radioactive compound, an ion and a smallmolecule.

[0100] In one embodiment, the membrane of the minicell comprises asystem for transferring a molecule from the interior of a minicell intothe cytoplasm of the cell. A representative system for transferring amolecule from the interior of a minicell into the cytoplasm of the cellis a Type III secretion system.

[0101] In one embodiment, the minicell further comprises a first andsecond nucleic acid, wherein the first nucleic acid comprises eukaryoticexpression sequences operably linked to a first ORF, and a secondnucleic acid, wherein the second nucleic acid comprises eubacterialexpression sequences operably linked to a second ORF. In one embodiment,one of the ORFs encodes a protein that comprises the binding moiety. Inone embodiment, the eubacterial expression sequences are induced and/orderepressed when the binding moiety is in contact with a target cell. Inone embodiment, the eukaryotic expression sequences are induced and/orderepressed when the nucleic acid is in the cytoplasm of a eukaryoticcell. In one embodiment, the protein encoded by the first ORF compriseseukaryotic secretion sequences and/or the protein encoded by the secondORF comprises eubacterial secretion sequences. In one embodiment, theligand is selected from the group consisting of a cytokine, hormone, anda small molecule.

[0102] In one aspect, the invention provides a minicell displaying asynthetic linking moiety, wherein the synthetic linking moiety iscovalenty or non-covalently attached to a membrane component of themincell.

[0103] In one aspect, the invention provides a sterically stabilizedminicell comprising a displayed moiety that has a longer half-life invivo than a wild-type minicell, wherein the displayed moiety is ahydrophilic polymer that comprises a PEG moiety, a carboxylic group of apolyalkylene glycol or PEG stearate.

[0104] In one aspect, the invention provides a minicell having amembrane comprising an exogenous lipid, wherein a minicell comprisingthe exogenous lipid has a longer half-life in vivo than a minicelllacking the exogenous lipid, and wherein the minicell is selected fromthe group consisting of a eubacterial minicell, a poroplast, aspheroplast and a protoplast. In one embodiment, the exogenous lipid isa derivitized lipid which may, by way of non-limiting example, bephosphatidylethanolamine derivatized with PEG, DSPE-PEG, PEG stearate;PEG-derivatized phospholipids, a PEG ceramide or DSPE-PEG.

[0105] In one embodiment, the exogenous lipid is not present in awild-type membrane, or is present in a different proportion than isfound in minicells comprising a wild-type membrane. The exogenous lipidcan be a ganglioside, sphingomyelin, monosialoganglioside GM1,galactocerebroside sulfate, 1,2-sn-dimyristoylphosphatidylcholine,phosphatidylinositol and cardiolipin.

[0106] In one embodiment, the linking moiety is non-covalently attachedto the minicell. In one embodiment, one of the linking moiety and themembrane component comprises biotin, and the other comprises avidin orstreptavidin. In one embodiment, the synthetic linking moiety is across-linker. In one embodiment, the cross-linker is a bifunctionalcross-linker.

[0107] In one aspect, the invention provides a method of transferring amembrane protein from a minicell membrane to a biological membranecomprising contacting a minicell to the biological membrane, wherein theminicell membrane comprises the membrane protein, and allowing themincell and the biological membrane to remain in contact for a period oftime sufficient for the transfer to occur.

[0108] In one embodiment, the biological membrane is a cytoplasmicmembrane or an organellar membrane. In one embodiment, the biologicalmembrane is a membrane selected from the group consisting of a membraneof a pathogen, a membrane of an infected cell and a membrane of ahyperproliferative cell. In one embodiment, the biological membrane isthe cytoplasmic membrane of a recipient cell, which may be a culturedcell and a cell within an organism. In one embodiment, the biologicalmembrane is present on a cell that has been removed from an animal, thecontacting occurs in vitro, after which the cell is returned to theorganism.

[0109] In one embodiment, the membrane protein is an enzyme. In thisembodiment, the membrane protein having enzymatic activity is selectedfrom the group consisting of a cytochrome P450 and a fusion protein, thefusion protein comprising a first polypeptide, the first polypeptidecomprising at least one polypeptide, wherein the second polypeptide hasenzymatic acitivity.

[0110] In one embodiment, the membrane protein is a membrane fusionprotein, the membrane fusion protein comprising a first polypeptide, thefirst polypeptide comprising at least one transmembrane domain or atleast one membrane anchoring domain; and a second polypeptide.

[0111] In one embodiment, the second polypeptide is a biologicallyactive polypeptide. In one embodiment, the minicell displays ligand or abinding moiety.

[0112] In one aspect, the invention provides a minicell that comprisesan expression construct comprising an ORF encoding a membrane proteinoperably linked to expression sequences, wherein the expressionsequences are induced and/or derepressed when the minicell is in contactwith a target cell.

[0113] In one embodiment, the biological membrane is a cytoplasmicmembrane or an organellar membrane. In one embodiment, the biologicalmembrane is a membrane selected from the group consisting of a membraneof a pathogen, a membrane of an infected cell and a membrane of ahyperproliferative cell. In one embodiment, the minicell displays aligand or a binding moiety selected from the group consisting of anantibody, an antibody derivative, an aptamer and a small molecule. Inone embodiment, the membrane protein is a membrane fusion protein, themembrane fusion protein comprising a first polypeptide, the firstpolypeptide comprising at least one transmembrane domain or at least onemembrane anchoring domain, and a second polypeptide. In one embodiment,the ligand is selected from the group consisting of a cytokine, hormone,and a small molecule.

[0114] In one aspect, the invention provides a pharmaceuticalcomposition comprising a minicell, wherein the minicell displays amembrane protein, wherein the membrane protein is selected from thegroup consisting of a eukaryotic membrane protein, an archeabacterialmembrane protein and an organellar membrane protein. In one embodiment,the membrane protein is selected from the group consisting of areceptor, a channel protein, a cellular adhesion factor and an integrin.In one embodiment, the pharmaceutical formulation further comprises anadjuvant. In one embodiment, the membrane protein comprises apolypeptide epitope displayed by a hyperproliferative cell. In oneembodiment, the membrane protein comprises an epitope displayed by aeukaryotic pathogen, an archeabacterial pathogen, a virus or an infectedcell.

[0115] In one aspect, the invention provides a pharmaceuticalcomposition comprising a minicell, wherein the minicell displays amembrane protein that is a fusion protein, the fusion protein comprising(i) a first polypeptide, the first polypeptide comprising at least onetransmembrane domain or at least one membrane anchoring domain; and (ii)a second polypeptide, wherein the second polypeptide is not derived froma eubacterial protein. In one embodiment, the pharmaceutical formulationfurther comprises an adjuvant. In one embodiment, the second polypeptidecomprises a polypeptide epitope displayed by a hyperproliferative cell.In one embodiment, the membrane protein comprises an epitope displayedby a eukaryotic pathogen, an archeabacterial pathogen, a virus or aninfected cell.

[0116] In one aspect, the invention provides a pharmaceuticalcomposition comprising a minicell, wherein the minicell displays amembrane conjugate, wherein the membrane conjugate comprises a membranecomponent chemically linked to a conjugated compound. In one embodiment,the membrane protein is selected from the group consisting of areceptor, a channel protein, a cellular adhesion factor and an integrin.In one embodiment, the pharmaceutical further comprises an adjuvant. Inone embodiment, the membrane component is a polypeptide comprising atleast one transmembrane domain or at least one membrane anchoringdomain, or a lipid that is part of a membrane. In one embodiment, theconjugated compound is a polypeptide, and the chemical linkage betweenthe membrane compound and the conjugated compound is not a peptide bond.In one embodiment, the conjugated compound is a nucleic acid. In oneembodiment, the conjugated compound is an organic compound. In oneembodiment, the organic compound is selected from the group consistingof a narcotic, a toxin, a venom, a sphingolipid and a soluble protein.

[0117] In one aspect, the invention provides a method of making apharmaceutical composition comprising a minicell, wherein the minicelldisplays a membrane protein, wherein the membrane protein is selectedfrom the group consisting of a eukaryotic membrane protein, anarcheabacterial membrane protein and an organellar membrane protein. Inone embodiment, the method further comprises adding an adjuvant to thepharmaceutical formulation. In one embodiment, the method furthercomprises desiccating the formulation. In one embodiment, the methodfurther comprises adding a suspension buffer to the formulation. In oneembodiment, the method further comprises making a chemical modificationof the membrane protein. In one embodiment, the chemical modification isselected from the group consisting of glycosylation, deglycosylation,phosphorylation, dephosphorylation and proteolysis. In one aspect, theinvention provides a method of making a pharmaceutical compositioncomprising a minicell, wherein the minicell displays a membrane proteinthat is a fusion protein, the fusion protein comprising (i) a firstpolypeptide, the first polypeptide comprising at least one transmembranedomain or at least one membrane anchoring domain; and (ii) a secondpolypeptide, wherein the second polypeptide is not derived from aeubacterial protein.

[0118] In one aspect, the invention provides a method of making apharmaceutical formulation comprising a minicell, wherein the minicelldisplays a membrane conjugate, wherein the membrane conjugate comprisesa membrane component chemically linked to a conjugated compound. In oneembodiment, the method further comprises adding an adjuvant to thepharmaceutical formulation. In one embodiment, the membrane component isa polypeptide comprising at least one transmembrane domain or at leastone membrane anchoring domain, or a lipid that is part of a membrane. Inone embodiment, the conjugated compound is a polypeptide, and thechemical linkage between the membrane compound and the conjugatedcompound is not a peptide bond. In one embodiment, the conjugatedcompound is a nucleic acid. In one embodiment, the conjugated compoundis an organic compound. In one embodiment, the organic compound isselected from the group consisting of a narcotic, a toxin, a venom, anda sphingolipid.

[0119] In one aspect, the invention provides a method of detecting anagent that is specifically bound by a binding moiety, comprisingcontacting a minicell displaying the binding moiety with a compositionknown or suspected to contain the agent, and detecting a signal that ismodulated by the binding of the agent to the binding moiety. In oneembodiment, the agent is associated with a disease. In one embodiment,the minicell comprises a detectable compound. In one embodiment, thebinding moiety is antibody or antibody derivative. In one embodiment,the composition is an environmental sample. In one embodiment, thecomposition is a biological sample. In one embodiment, the biologicalsample is selected from the group consisting of blood, serum, plasma,urine, saliva, a biopsy sample, feces and a skin patch.

[0120] In one aspect, the invention provides a method of in situ imagingof a tissue or organ, comprising administering to an organism a minicellcomprising an imaging agent and a binding moiety and detecting theimaging agent in the organism.

[0121] In one embodiment, the minicell is a eubacterial minicell, aporoplast, a spheroplast or a protoplast. In one embodiment, the bindingmoiety is an antibody or antibody derivative. In one embodiment, thebinding moiety specifically binds a cell surface antigen. In oneembodiment, the cell surface antigen is an antigen displayed by atumorigenic cell, a cancer cell, and an infected cell. In oneembodiment, the cell surface antigen is a tissue-specific antigen. Inone embodiment, the method of imaging is selected from the groupconsisting of magnetic resonance imaging, ultrasound imaging; andcomputer axaial tomography (CAT). In one aspect, the invention providesa device comprising a microchip operatively associated with a biosensorcomprising a minicell, wherein the microchip comprises or contacts theminicell, and wherein the minicell displays a binding moiety.

[0122] In one embodiment, the invention provides a method of detecting asubstance that is specifically bound by a binding moiety, comprisingcontacting the device of claim 16 with a composition known or suspectedto contain the substance, and detecting a signal from the device,wherein the signal changes as a function of the amount of the substancepresent in the composition. In one embodiment, the composition is abiological sample or an environmental sample.

[0123] In one aspect, the invention provides a method of identifying anagent that specifically binds a target compound, comprising contacting aminicell displaying the target compound with a library of compounds, andidentifying an agent in the library that binds the target compound. Inone embodiment, the library of compounds is a protein library. In oneembodiment, the protein library is selected from the group consisting ofa phage display library, a phagemid display library, a baculoviruslibrary, a yeast display library, and a ribosomal display library. Inone embodiment, the library of compounds is selected from the groupconsisting of a library of aptamers, a library of synthetic peptides anda library of small molecules.

[0124] In one embodiment, the target compound is a target polypeptide.In one embodiment, the minicell comprises an expression constructcomprising expression sequences operably linked to an ORF encoding thetarget polypeptide. In one embodiment, the target polypeptide is amembrane protein. In one embodiment, the membrane protein is a receptoror a channel protein. In one embodiment, the membrane protein is anenzyme. In one embodiment, the target compound is a membrane fusionprotein, the membrane fusion protein comprising a first polypeptide,wherein the first polypeptide comprises at least one transmembranedomain or at least one membrane anchoring domain; and a secondpolypeptide, wherein the second polypeptide comprises amino acidsequences derived from a target polypeptide. In one embodiment, themethod further comprises comparing the activity of the target compoundin the presence of the agent to the activity of the target compound inthe absence of the agent.

[0125] In one embodiment, the activity of the target compound is anenzyme activity. In one embodiment, the activity of the target compoundis a binding activity. In one embodiment, the invention furthercomprises comparing the binding of the agent to the target compound tothe binding of a known ligand of the target compound. In one embodiment,a competition assay is used for the comparing.

[0126] In one aspect, the invention provides a device comprisingmicrochips operatively associated with a biosensor comprising a set ofminicells in a prearranged pattern, wherein the each of the microchipscomprise or contact a minicell, wherein each of the minicell displays adifferent target compound, and wherein binding of a ligand to a targetcompound results in an increased or decreased signal. In one embodiment,the invention provides a method of identifying an agent thatspecifically binds a target compound, comprising contacting the devicewith a library of compounds, and detecting a signal from the device,wherein the signal changes as a function of the binding of an agent tothe target compound. In one embodiment, the invention provides a methodof identifying an agent that specifically blocks the binding of a targetcompound to its ligand, comprising contacting the device with a libraryof compounds, and detecting a signal from the device, wherein the signalchanges as a function of the binding of an agent to the target compound.

[0127] In one aspect, the invention provides a method of making aantibody that specifically binds a protein domain, wherein the domain isin its native conformation, wherein the domain is contained within aprotein displayed on a minicell, comprising contacting the minicell witha cell, wherein the cell is competent for producing antibodies to anantigen contacted with the cell, in order to generate an immunogenicresponse in which the cell produces the antibody.

[0128] In one embodiment, the protein displayed on a minicell is amembrane protein. In one embodiment, the membrane protein is a receptoror a channel protein. In one embodiment, the domain is found within thesecond polypeptide of a membrane fusion protein, wherein the membranefusion protein comprises a first polypeptide, wherein the firstpolypeptide comprises at least one transmembrane domain or at least onemembrane anchoring domain. In one embodiment, the contacting occurs invivo. In one embodiment, the antibody is a polyclonal antibody or amonoclonal antibody. In one embodiment, the contacting occurs in ananimal that comprises an adjuvant.

[0129] In one aspect, the invention provides the method of making anantibody derivative that specifically binds a protein domain, whereinthe domain is in its native conformation, wherein the domain isdisplayed on a minicell, comprising contacting the minicell with aprotein library, and identifying an antibody derivative from the proteinlibrary that specifically binds the protein domain. In one embodiment,the protein library is selected from the group consisting of a phagedisplay library, a phagemid display library, and a ribosomal displaylibrary.

[0130] In one aspect, the invention provides a method of making anantibody or antibody derivative that specifically binds an epitope,wherein the epitope is selected from the group consisting of (i) anepitope composed of amino acids found within a membrane protein, (ii) anepitope present in an interface between a membrane protein and amembrane component, (iii) an epitope present in an interface between amembrane protein and one or more other proteins and (iv) an epitope in afusion protein, the fusion protein comprising a first polypeptide, thefirst polypeptide comprising at least one transmembrane domain or atleast one membrane anchoring domain, and a second polypeptide, thesecond polypeptide comprising the epitope; comprising contacting aminicell displaying the epitope with a protein library, or to a cell,wherein the cell is competent for producing antibodies to an antigencontacted with the cell, in order to generate an immunogenic response inwhich the cell produces the antibody.

[0131] In one embodiment, the cell is contacted in vivo. In variousembodiments, the antibody is a polyclonal antibody or a monoclonalantibody. In one embodiment, the protein library is contacted in vitro.In one embodiment, the protein library is selected from the groupconsisting of a phage display library, a phagemid display library, and aribosomal display library.

[0132] In one aspect, the invention provides a method of determining therate of transfer of nucleic acid from a minicell to a cell, comprising(a) contacting the cell to the minicell, wherein the minicell comprisesthe nucleic acid, for a measured period of time; (b) separatingminicells from the cells; (c) measuring the amount of nucleic acid inthe cells,wherein the amount of nucleic acid in the cells over the setperiod of time is the rate of transfer of a nucleic acid from aminicell.

[0133] In one aspect, the invention provides a method of determining theamount of a nucleic acid transferred to a cell from a minicell,comprising (a) contacting the cell to the minicell, wherein the minicellcomprises an expression element having eukaryotic expression sequencesoperably linked to an ORF encoding a detectable polypeptide, wherein theminicell displays a binding moiety, and wherein the binding moiety bindsan epitope of the cell; and (b) detecting a signal from the detectablepolypeptide, wherein a change in the signal corresponds to an increasein the amount of a nucleic acid transferred to a cell.

[0134] In one embodiment, the cell is a eukaryotic cell. By way ofnon-limiting example, a eukaryotic cell can be a plant cell, a fungalcell, a unicellular eukaryote, an animal cell, a mammalian cell, a ratcell, a mouse cell, a primate cell or a human cell.

[0135] In one embodiment, the binding moiety is an antibody or antibodyderivative. In one embodiment, the binding moiety is a single-chainantibody. In one embodiment, the binding moiety is an aptamer. In oneembodiment, the binding moiety is an organic compound. In oneembodiment, the detectable polypeptide is a fluorescent polypeptide.

[0136] In one aspect, the invention provides a method of detecting theexpression of an expression element in a cell, comprising (a) contactingthe cell to a minicell, wherein the minicell comprises an expressionelement having cellular expression sequences operably linked to an ORFencoding a detectable polypeptide, wherein the minicell displays abinding moiety, and wherein the binding moiety binds an epitope of thecell; (b) incubating the cell and the minicell for a period of timeeffective for transfer of nucleic acid from the minicell to the cell;and (c) detecting a signal from the detectable polypeptide, wherein anincrease in the signal corresponds to an increase in the expression ofthe expression element.

[0137] In one embodiment, the cell is a eukaryotic cell and theexpression sequences are eukaryotic expression sequences. In oneembodiment, the eukaryotic cell is a mammalian cell. In one embodiment,the binding moiety is an antibody or antibody derivative. In oneembodiment, the binding moiety is a single-chain antibody. In oneembodiment, the binding moiety is an aptamer. In one embodiment, thebinding moiety is an organic compound.

[0138] In a related aspect, the invention provides methods of detectingthe transfer of a fusion protein from the cytosol to an organelle of aeukaryotic cell, comprising (a) contacting the cell to a minicell,wherein (i) the minicell comprises an expression element havingeukaryotic expression sequences operably linked to an ORF encoding afusion protein, wherein the fusion protein comprises a first polypeptidethat comprises organellar delivery sequences, and a second polypeptidethat comprises a detectable polypeptide; and (ii) the minicell displaysa binding moiety that binds an epitope of the cell, or an epitope of anorganelle; (b) incubating the cell and the minicell for a period of timeeffective for transfer of nucleic acid from the minicell to the cell andproduction of the fusion protein; and (c) detecting a signal from thedetectable polypeptide, wherein a change in the signal corresponds to anincrease in the amount of the fusion protein transferred to theorganelle.

[0139] In one aspect, the invention provides a minicell comprising atleast one nucleic acid, wherein the minicell displays a binding moietydirected to a target compound, wherein the binding moiety is selectedfrom the group consisting of (i) a eukaryotic membrane protein; (ii) anarcheabacterial membrane protein; (iii) an organellar membrane protein;and (iv) a fusion protein, the fusion protein comprising a firstpolypeptide, the first polypeptide comprising at least one transmembranedomain or at least one membrane anchoring domain; and a secondpolypeptide, wherein the second polypeptide is not derived from aeubacterial protein and is neither a His tag nor aglutathione-S-transferase polypeptide, and wherein the polypeptidecomprises a binding moiety.

[0140] In one embodiment, the nucleic acid comprises an expressionconstruct comprising expression sequences operably linked to an ORFencoding a protein selected from the group consisting of (i) theeukaryotic membrane protein, (ii) the archeabacterial membrane protein,(iii) the organellar membrane protein; and (iv) the fusion protein.

[0141] In one embodiment, the nucleic acid comprises an expressionconstruct comprising expression sequences operably linked to an ORF,wherein the ORF encodes a therapeutic polypeptide. In one embodiment,the therapeutic polypeptide is a membrane polypeptide. In oneembodiment, the therapeutic polypeptide is a soluble polypeptide. In oneembodiment, the soluble polypeptide comprises a cellular secretionsequence. In one embodiment, the expression sequences are inducibleand/or repressible.

[0142] In one embodiment, the expression sequences are induced and/orderepressed when the binding moiety displayed by the minicell binds toits target compound. In one embodiment, the nucleic acid comprises anexpression construct comprising expression sequences operably linked toan ORF, wherein the ORF encodes a polypeptide having an amino acidsequence that facilitates cellular transfer of a biologically activecompound contained within or displayed by the minicell. In oneembodiment, the membrane of the minicell comprises a system fortransferring a molecule from the interior of a minicell into thecytoplasm of the cell. In one embodiment, the system for transferring amolecule from the interior of a minicell into the cytoplasm of the cellis a Type III secretion system.

[0143] In one aspect, the invention provides a method of introducing anucleic acid into a cell, comprising contacting the cell with a minicellthat comprises the nucleic acid, wherein the minicell displays a bindingmoiety, wherein the binding moiety is selected from the group consistingof (i) a eukaryotic membrane protein; (ii) an archeabacterial membraneprotein; (iii) an organellar membrane protein; and (iv) a fusionprotein, the fusion protein comprising a first polypeptide, the firstpolypeptide comprising at least one transmembrane domain or at least onemembrane anchoring domain; and a second polypeptide, wherein the secondpolypeptide is not derived from a eubacterial protein and is neither aHis tag nor a glutathione-S-transferase polypeptide, and wherein thepolypeptide comprises a binding moiety; and wherein the binding moietybinds an epitope of the cell.

[0144] In one embodiment, the nucleic acid comprises an expressionconstruct comprising expression sequences operably linked to an ORFencoding a protein selected from the group consisting of (i) theeukaryotic membrane protein, (ii) the archeabacterial membrane protein,(iii) the organellar membrane protein; and (iv) a fusion protein.

[0145] In one embodiment, the nucleic acid comprises an expressionconstruct comprising expression sequences operably linked to an ORF,wherein the ORF encodes a therapeutic polypeptide. In one embodiment,the expression sequences are inducible and/or derepressible. In oneembodiment, the expression sequences are induced or derepressed when thebinding moiety displayed by the minicell binds its target compound. Inone embodiment, the expression sequences are induced or derepressed by atransactivation or transrepression event. In one embodiment, the nucleicacid comprises an expression construct comprising expression sequencesoperably linked to an ORF, wherein the ORF encodes a polypeptide havingan amino acid sequence that facilitates cellular transfer of abiologically active compound contained within or displayed by theminicell.

[0146] In one aspect, the invention provides a minicell comprising anucleic acid, wherein the nucleic acid comprises eukaryotic expressionsequences and eubacterial expression sequences, each of which isindependently operably linked to an ORF.

[0147] In one embodiment, the minicell displays a binding moiety. In oneembodiment, the eubacterial expression sequences are induced and/orderepressed when the binding moiety is in contact with a target cell. Inone embodiment, the eukaryotic expression sequences are induced and/orderepressed when the nucleic acid is in the cytoplasm of a eukaryoticcell. In one embodiment, the protein encoded by the ORF compriseseubacterial or eukaryotic secretion sequences.

[0148] In one aspect, the invention provides a minicell comprising afirst and second nucleic acid, wherein the first nucleic acid compriseseukaryotic expression sequences operably linked to a first ORF, and asecond nucleic acid, wherein the second nucleic acid compriseseubacterial expression sequences operably linked to a second ORF.

[0149] In one embodiment, the minicell displays a binding moiety. In oneembodiment, the eubacterial expression sequences are induced and/orderepressed when the binding moiety is in contact with a target cell. Inone embodiment, the eukaryotic expression sequences are induced and/orderepressed when the nucleic acid is in the cytoplasm of a eukaryoticcell. In one embodiment, the protein encoded by the first ORF compriseseukaryotic secretion sequences and/or the protein encoded by the secondORF comprises eubacterial secretion sequences.

[0150] In one aspect, the invention provides a method of introducinginto and expressing a nucleic acid in an organism, comprising contactinga minicell to a cell of the organism, wherein the minicell comprises thenucleic acid.

[0151] In one embodiment, the minicell displays a binding moiety. In oneembodiment, the nucleic acid comprises a eukaryotic expressionconstruct, wherein the eukaryotic expression construct compriseseukaryotic expression sequences operably linked to an ORF. In oneembodiment, the ORF encodes a protein selected from the group consistingof a membrane protein, a soluble protein and a protein comprisingeukaryotic secretion signal sequences. In one embodiment, the nucleicacid comprises a eubacterial expression construct, wherein theeubacterial expression construct comprises eubacterial expressionsequences operably linked to an ORF. In one embodiment, the minicelldisplays a binding moiety, wherein the eubacterial expression sequencesare induced and/or derepressed when the binding moiety is in contactwith a target cell. In one embodiment, the protein encoded by the ORFcomprises eubacterial secretion sequences. In one aspect, the inventionprovides a minicell comprising a crystal of a membrane protein. In oneembodiment, the minicell is a eubacterial minicell, a poroplast, aspheroplast or a protoplast. In one embodiment, the membrane protein isa receptor. In one embodiment, the receptor is a G-protein coupledreceptor. In one embodiment, the crystal is displayed.

[0152] In a related aspect, the invention provides a minicell membranepreparation comprising a crystal of a membrane protein.

[0153] In one embodiment, the membrane protein is a fusion protein, thefusion protein comprising a first polypeptide, the first polypeptidecomprising at least one transmembrane domain or at least one membraneanchoring domain, and a second polypeptide. In one embodiment, thecrystal is a crystal of the second polypeptide. In one embodiment, thecrystal is displayed.

[0154] In one aspect, the invention provides a method of determining thethree-dimensional structure of a membrane protein, comprising preparinga crystal of the membrane protein in a minicell, and determining thethree-dimensional structure of the crystal.

[0155] In one aspect, the invention provides a method for identifyingligand-interacting atoms in a defined three-dimensional structure of atarget protein, comprising (a) preparing one or more variant proteins ofa target protein having a known or predicted three-dimensionalstructure, wherein the target protein binds a preselected ligand; (b)expressing and displaying a variant protein in a minicell; and (c)determining if a minicell displaying the variant protein binds thepreselected ligand with increased or decreased affinity as compared tothe binding of the preselected ligand to the target protein.

[0156] In one embodiment, the ligand is a protein that forms a multimerwith the target protein, and the ligand interacting atoms are atoms inthe defined three-dimensional structure are atoms that are involved inprotein-protein interactions. In one embodiment, the ligand is acompound that induces a conformational change in the target protein, andthe defined three-dimensional structure is the site of theconformational change. In one embodiment, the method for identifyingligands of a target protein, further comprising identifying the chemicaldifferences in the variant proteins as compared to the target protein.In one embodiment, the invention further comprises mapping the chemicaldifferences onto the defined three-dimensional structure, andcorrelating the effect of the chemical differences on the definedthree-dimensional structure. In one embodiment, the target protein is awild-type protein. In one aspect, the invention provides a minicelllibrary, comprising two or more minicells, wherein each minicellcomprises a different exogenous protein. In one embodiment, the minicellis a eubacterial minicell, a poroplast, a spheroplast or a protoplast.In one embodiment, the exogenous protein is a displayed protein. In oneembodiment, the exogenous protein is a membrane protein. In oneembodiment, the membrane protein is a receptor. In one embodiment, theprotein is a soluble protein that is contained within or secreted fromthe minicell. In one embodiment, minicells within the library comprisean expression element that comprises expression sequences operablylinked to a nucleic acid having an ORF that encodes the exogenousprotein. In one embodiment, the nucleic acid has been mutagenized; themutagenesis can be site-directed or random. In one embodiment, an activesite of the exogenous protein has a known or predicted three-dimensionalstructure, and the a portion of the ORF encoding the active site hasbeen mutagenized. In one embodiment, each of the minicells comprises anexogenous protein that is a variant of a protein having a known orpredicted three-dimensional structure.

[0157] In one aspect, the invention provides a minicell library,comprising two or more minicells, wherein each minicell comprises adifferent fusion protein, each of the fusion protein comprising a firstpolypeptide that is a constant polypeptide, wherein the constantpolypeptide comprises at least one transmembrane domain or at least onemembrane anchoring domain, and a second polypeptide, wherein the secondpolypeptide is a variable amino acid sequence that is different in eachfusion proteins. In one embodiment, minicells within the librarycomprise an expression element that comprises expression sequencesoperably linked to a nucleic acid having an ORF that encodes the fusionprotein. In one embodiment, the second polypeptide of the fusion proteinis encoded by a nucleic acid that has been cloned. In one embodiment,each of the second polypeptide of each of the fusion proteins comprisesa variant of an amino acid sequence from a protein having a known orpredicted three-dimensional structure.

[0158] In one aspect, the invention provides a minicell library,comprising two or more minicells, wherein each minicell comprises aconstant protein that is present in each minicell and a variable proteinthat differs from minicell to minicell. In one embodiment, one of theconstant and variable proteins is a receptor, and the other of theconstant and variable proteins is a co-receptor. In one embodiment, eachof the constant and variable proteins is different from each other andis a factor in a signal transduction pathway. In one embodiment, one ofthe constant and variable proteins is a G-protein, and the other of theconstant and variable proteins is a G-protein coupled receptor.

[0159] In one embodiment, one of the constant and variable proteinscomprises a first transrepression domain, and the other of the constantand variable comprises a second transrepression domain, wherein thetransrepression domains limit or block expression of a reporter genewhen the constant and variable proteins associate with each other.

[0160] In one embodiment, one of the constant and variable proteinscomprises a first transactivation domain, and the other of the constantand variable comprises a second transactivation domain, wherein thetransactivation domains stimulate expression of a reporter gene when theconstant and variable proteins associate with each other.

[0161] In one aspect, the invention provides a method of identifying anucleic acid that encodes a protein that binds to or chemically alters apreselected ligand, comprising (a) separately contacting the ligand withindividual members of a minicell library, wherein minicells in thelibrary comprise expression elements, wherein the expression elementscomprise DNA inserts, wherein an ORF in the DNA insert is operablylinked to expression sequences, in order to generate a series ofreaction mixes, each reaction mix comprising a different member of theminicell library; (b) incubating the reaction mixes, thereby allowing aprotein that binds to or chemically alters the preselected ligand tobind or chemically alter the preselected ligand; (c) detecting a changein a signal from reaction mixes in which the ligand has been bound orchemically altered; (d) preparing DNA from reaction mixes in which theligand has been bound or chemically altered; wherein the DNA is anucleic acid that encodes a protein that binds to or chemically altersthe preselected ligand.

[0162] In one embodiment, the minicell is a eubacterial minicell, aporoplast, a spheroplast or a protoplast. In one embodiment, thepreselected ligand is a biologically active compound. In one embodiment,the preselected ligand is a therapeutic drug. In one embodiment, aprotein that binds or chemically alters the preselected ligand is atarget protein for compounds that are therapeutic for a disease that istreated by administering the drug to an organism in need thereof. In oneembodiment, the preselected ligand is detectably labeled, the mincellcomprises a detectable compound, and/or a chemically altered derivativeof the protein is detectably labeled.

[0163] In one aspect, the invention provides a method of determining theamino acid sequence of a protein that binds or chemically alters apreselected ligand, comprising: (a) contacting the ligand with aminicell library, wherein minicells in the library comprise expressionelements, wherein the expression elements comprise DNA inserts, whereinan ORF in the DNA insert is operably linked to expression sequences; (b)incubating the mixture of ligand and minicells, under conditions whichallow complexes comprising ligands and minicells to form and/or chemicalreactions to occur; (c) isolating or identifying the complexes from theligand and the mixture of ligand and minicells; (d) preparing DNA froman expression element found in one or more of the complexes, or in aminicell thereof; (e) determining the nucleotide sequence of the ORF inthe DNA; and (f) generating an amino sequence by in silicon translation,wherein the amino acid sequence is or is derived from a protein thatbinds or chemically alters a preselected ligand.

[0164] In one embodiment, the minicell is a eubacterial minicell, aporoplast, a spheroplast or a protoplast. In one embodiment, the DNA isprepared by isolating DNA from the complexes, or in a minicell thereof.In one embodiment, the DNA is prepared by amplifying DNA from thecomplexes, or in a minicell thereof. In one embodiment, the protein is afusion protein. In one embodiment, the protein is a membrane or asoluble protein. In one embodiment, the protein comprises secretionsequences. In one embodiment, the preselected ligand is a biologicallyactive compound. In one embodiment, the preselected ligand is atherapeutic drug. In one embodiment, the preselected ligand is atherapeutic drug, and the protein that binds the preselected ligand is atarget protein for compounds that are therapeutic for a disease that istreated by administering the drug to an organism in need thereof.

[0165] In one aspect, the invention provides a method of identifying anucleic acid that encodes a protein that inhibits or blocks an agentfrom binding to or chemically altering a preselected ligand, comprising:(a) separately contacting the ligand with individual members of aminicell library, wherein minicells in the library comprise expressionelements, wherein the expression elements comprise DNA inserts, whereinan ORF in the DNA insert is operably linked to expression sequences, inorder to generate a series of reaction mixes, each reaction mixcomprising a different member of the minicell library; (b) incubatingthe reaction mixes, thereby allowing a protein that binds to orchemically alters the preselected ligand to bind or chemically alter thepreselected ligand; (c) detecting a change in a signal from reactionmixes in which the ligand has been bound or chemically altered; (d)preparing DNA from reaction mixes in which the change in signal ligandhas been bound or chemically altered; wherein the DNA is a nucleic acidthat encodes a protein that inhibits or blocks the agent from binding toor chemically altering the preselected ligand

[0166] In one embodiment, the minicell is a eubacterial minicell, aporoplast, a spheroplast or a protoplast. In one embodiment, the DNA hasa nucleotide sequence that encodes the amino acid sequence of theprotein that inhibits or blocks the agent from binding to or chemicallyaltering the preselected ligand. In one embodiment, a protein that bindsor chemically alters the preselected ligand is a target protein forcompounds that are therapeutic for a disease that is treated byadministering the drug to an organism in need thereof.

[0167] In one aspect, the invention provides a method of identifying anagent that effects the activity of a protein, comprising contacting alibrary of two or more candidate agents with a minicell comprising theprotein or a polypeptide derived from the protein, assaying the effectof candidate agents on the activity of the protein, and identifyingagents that effect the activity of the protein.

[0168] In one embodiment, the protein or the polypeptide derived fromthe protein is displayed on the surface of the minicell. In oneembodiment, the protein is a membrane protein. In one embodiment, themembrane protein is selected from the group consisting of a receptor, achannel protein and an enzyme. In one embodiment, the activity of aprotein is a binding activity or an enzymatic activity. In oneembodiment, the library of compounds is a protein library. In oneembodiment, the protein library is selected from the group consisting ofa phage display library, a phagemid display library, and a ribosomaldisplay library. In one embodiment, the library of compounds is alibrary of aptamers. In one embodiment, the library of compounds is alibrary of small molecules.

[0169] In one aspect, the invention provides a method of identifying anagent that effects the activity of a protein domain containing a libraryof two or more candidate agents with a minicell displaying a membranefusion protein, the fusion protein comprising a first polypeptide, thefirst polypeptide comprising at least one transmembrane domain or atleast one membrane anchoring domain, and a second polypeptide, whereinthe second polypeptide comprises the protein domain.

[0170] In one aspect, the invention provides a method of identifyingundesirable side-effects of a biologically active compound that occur asa result of binding of the compound to a protein, wherein binding acompound to the protein is known to result in undesirable side effects,comprising contacting a minicell that comprises the protein to thebiologically active compound. In one embodiment, the invention providescomprises characterizing the binding of the biologically active compoundto the protein. In one embodiment, the invention provides comprisescharacterizing the effect of the biologically active compound on theactivity of the protein.

[0171] In one aspect, the invention provides a method for identifying anagent that effects the interaction of a first signaling protein with asecond signaling protein, comprising (a) contacting a library ofcompounds with a minicell, wherein the minicell comprises: (i) a firstprotein comprising the first signaling protein and a first trans-actingregulatory domain; (ii) a second protein comprising the second signalingprotein and a second trans-acting regulatory domain; and (iii) areporter gene, the expression of which is modulated by the interactionbetween the first trans-acting regulatory domain and the secondtrans-acting regulatory domain; and (b) detecting the gene product ofthe reporter gene.

[0172] In one embodiment, the trans-acting regulatory domains aretransactivation domains. In one embodiment, the trans-acting regulatorydomains are transrepression domains.

[0173] In one embodiment, the reporter gene is induced by theinteraction of the first trans-acting regulatory domain and the secondtrans-acting regulatory domain. In one embodiment, the agent thateffects the interaction of the first signaling protein with the secondsignaling protein is an agent that causes or promotes the interaction.In one embodiment, the reporter gene is repressed by the interaction ofthe first trans-acting regulatory domain and the second trans-actingregulatory domain. In one embodiment, the agent that effects theinteraction of the first signaling protein with the second signalingprotein is an agent that inhibits or blocks the interaction.

[0174] In one embodiment, the first signaling protein is a GPCR. In oneembodiment, the GPCR is an Edg receptor or a ScAMPER.

[0175] In one embodiment, the second signalling protein is a G-protein.In related embodiments, G-protein is selected from the group consistingof G-alpha-i, G-alpha-s, G-alpha-q, G-alpha-12/13 and Go. In oneembodiment, the library of compounds is a protein library. In oneembodiment, the protein library is selected from the group consisting ofa phage display library, a phagemid display library, and a ribosomaldisplay library. In one embodiment, the library of compounds is alibrary of aptamers. In one embodiment, the library of compounds is alibrary of small molecules.

[0176] In one aspect, the invention provides a method for identifying anagent that effects the interaction of a first signaling protein with asecond signaling protein, comprising contacting a library of two or morecandidate agents with a minicell, wherein the minicell comprises (a) afirst fusion protein comprising the first signaling protein and a firstdetectable domain; and (b) a second fusion protein comprising the secondsignaling protein and a second detectable domain, wherein a signal isgenerated when the first and second signaling proteins are in closeproximity to each other, and detecting the signal.

[0177] In one embodiment, the signal is fluorescence. In one embodiment,the first detectable domain and the second detectable domain arefluorescent and the signal is generated by FRET. In one embodiment, thefirst and second detectable domains are independently selected from thegroup consisting of a green fluorescent protein, a blue-shifted greenfluorescent protein, a cyan-shifted green fluorescent protein; ared-shifted green fluorescent protein; a yellow-shifted greenfluorescent protein, and a red fluorescent protein, wherein the firstfluorescent domain and the second fluorescent domain are not identical.

[0178] In one aspect, the invention provides a method of bioremediation,the method comprising contacting a composition that comprises anundesirable substance with a minicell, wherein the minicell alters thechemical structure and/or binds the undesirable substance.

[0179] In one aspect, the invention provides a method of bioremediation,the method comprising contacting a composition that comprises anundesirable substance with a minicell, wherein the mincell comprises anagent that alters the chemical structure of the undesirable substance.In one embodiment, the agent that alters the chemical structure of theundesirable substance is an inorganic catalyst. In one embodiment, theagent that alters the chemical structure of the undesirable substance isan enzyme. In one embodiment, the enzyme is a soluble protein containedwithin the minicell. In one embodiment, the enzyme is a secretedprotein. In one embodiment, the enzyme is a membrane protein. In oneembodiment, the membrane enzyme is selected from the group consisting ofa cytochrome P450, an oxidoreductase, a transferase, a hydrolase, alyase, an isomerase, a ligase and a synthetase. In one embodiment, theagent that alters the chemical structure of the undesirable substance isa fusion protein comprising a first polypeptide that comprises atransmembrane domain or at least one membrane-anchoring domain, and asecond polypeptide, wherein the second polypeptide is an enzyme moiety.

[0180] In one aspect, the invention provides a method of bioremediation,the method comprising contacting a composition that comprises anundesirable substance with a minicell, wherein the mincell comprises anagent that binds an undesirable substance. In one embodiment, theundesirable substance binds to and is internalized by the minicell or isotherwise inactivated by selective absorption. In one embodiment, theagent that binds the undesirable substance is a secreted solubleprotein. In one embodiment, the secreted protein is a transportaccessory protein. In one embodiment, the agent that binds theundesirable substance is a membrane protein. In one embodiment, theundesirable substance is selected from the group consisting of a toxin,a pollutant and a pathogen. In one embodiment, the agent that binds theundesirable substance is a fusion protein comprising a first polypeptidethat comprises a transmembrane domain or at least one membrane-anchoringdomain, and a second polypeptide, wherein the second polypeptide is abinding moiety. In one embodiment, wherein the binding moiety isselected from the group consisting of an antibody, an antibodyderivative, the active site of a non-enzymatically active mutant enzyme,a single-chain antibody and an aptamer.

[0181] In one aspect, the invention provides a minicell-producing parentcell, wherein the parent cell comprises one or more of the following (a)an expression element that comprises a gene operably linked toexpression sequences that are inducible and/or repressible, whereininduction or repression of the gene regulates the copy number of anepisomal expression construct; (b) a mutation in an endogenous gene,wherein the mutation regulates the copy number of an episomal expressionconstruct; (c) an expression element that comprises a gene operablylinked to expression sequences that are inducible and/or repressible,wherein induction or repression of the gene causes or enhances theproduction of minicells; and (d) a mutation in an endogenous gene,wherein the mutation causes or enhances minicell production.

[0182] In one embodiment, the invention comprises an episomal expressionconstruct. In one embodiment, the invention further comprises achromosomal expression construct. In one embodiment, the expressionsequences of the expression construct are inducible and/or repressible.In one embodiment, the minicell-producing parent cell comprises abiologically active compound. In one embodiment, the gene that causes orenhances the production of minicells has a gene product that is involvedin or regulates DNA replication, cellular division, cellularpartitioning, septation, transcription, translation, or protein folding.

[0183] In one aspect, the invention provides a minicell-producing parentcell, wherein the parent cell comprises an expression construct, whereinthe expression construct comprises expression sequences operably linkedto an ORF that encodes a protein, and a regulatory expression element,wherein the regulatory expression element comprises expression sequencesoperably linked to a regulatory gene that encodes a factor thatregulates the expression of the ORF. In one embodiment, the expressionsequences of the expression construct are inducible and/or repressible.In one embodiment, the expression sequences of the regulatory expressionconstruct are inducible and/or repressible. In one embodiment, one ormore of the expression element or the regulatory expression element islocated on a chromosome of the parent cell. In one embodiment, one ormore of the expression element or the regulatory expression element islocated on an episomal expression construct. In one embodiment, both ofthe expression element and the regulatory expression element are locatedon an episomal expression construct, and one or both of the expressionelement and the regulatory expression element segregates into minicellsproduced from the parent cell. In one embodiment, the minicell-producingparent cell comprises a biologically active compound. In one embodiment,the biologically active compound segregates into minicells produced fromthe parent cell. In one embodiment, the ORF encodes a membrane proteinor a soluble protein. In one embodiment, the protein comprises secretionsequences. In one embodiment, the gene product of the gene regulates theexpression of the ORF. In one embodiment, the gene product is atranscription factor. In one embodiment, the gene product is a RNApolymerase. In one embodiment, the parent cell is MC-T7.

[0184] In one aspect, the invention provides a minicell comprising abiologically active compound, wherein the minicell displays a bindingmoiety, wherein the minicell selectively absorbs and/or internalizes anundesirable compound, and the minicell is a poroplast, spheroplast orprotoplast. In one embodiment, the binding moiety is selected from thegroup consisting of an antibody, an antibody derivative, a receptor andan active site of a non-catalytic derivative of an enzyme. In oneembodiment, the binding moiety is a single-chain antibody. In oneembodiment, the binding moiety is directed to a ligand selected from thegroup consisting of an epitope displayed on a pathogen, an epitopedisplayed on an infected cell and an epitope displayed on ahyperproliferative cell. In one embodiment, the biologically activecompound is selected from the group consisting of a radioisotope, apolypeptide, a nucleic acid and a small molecule. In one embodiment, aligand binds to and is internalized by the minicell or is otherwiseinactivated by selective absorption. In one embodiment, the inventionprovides a pharmaceutical composition comprising the minicell. In oneaspect, the invention provides a method of reducing the freeconcentration of a substance in a composition, wherein the substancedisplays a ligand specifically recognized by a binding moiety,comprising contacting the composition with a minicell that displays thebinding moiety, wherein the binding moiety binds the substance, therebyreducing the free concentration of the substance in the composition. Inone embodiment, the substance is selected from the group consisting of anucleic acid, a lipid, a polypeptide, a radioactive compound, an ion anda small molecule. In one embodiment, the binding moiety is selected fromthe group consisting of an antibody, an antibody derivative, a channelprotein and a receptor.

[0185] In one embodiment, the composition is present in an environmentincluding but not limited to water, air or soil. In one embodiment, thecomposition is a biological sample from an organism, including but notlimited to blood, serum, plasma, urine, saliva, a biopsy sample, feces,tissue and a skin patch. In one embodiment, the substance binds to andis internalized by the minicell or is otherwise inactivated by selectiveabsorption. In one embodiment, the biological sample is returned to theorganism after being contacting to the minicell.

[0186] For a better understanding of the present invention, reference ismade to the accompanying detailed description and its scope will bepointed out in the appended claims. All references cited herein arehereby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0187]FIG. 1 is a Western blot in which Edg-1-6×His and Edg-3-6×Hisproteins expressed in minicells produced from MC-T7 cells.

[0188]FIG. 2 shows induction of MalE(L)-NTR in isolated minicells.

ABBREVIATIONS AND DEFINITIONS

[0189] For brevity's sake, the single-letter amino acid abbreviationsare used in some instances herein. Table 1 describes the correspondencebetween the 1- and 3-letter amino acid abbreviations. TABLE 1 THREE- ANDONE-LETTER ABBREVIATIONS FOR AMINO ACIDS Three-letter One-letter Aminoacid abbreviation symbol Alanine Ala A Arginine Arg R Asparagine Asn NAspartic Acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu EGlycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine LysK Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0190] A “conjugatable compound” or “attachable compound” is capable ofbeing attached to another compound. The terms “conjugated to” and“cross-linked with” indicate that the conjugatable compound is in thestate of being attached to another compound.A “conjugate” is thecompound formed by the attachment of a conjugatable compound orconjugatable moiety to another compound.

[0191] “Culturing” signifies incubating a cell or organism underconditions wherein the cell or organism can carry out some, if not all,biological processes. For example, a cell that is cultured may begrowing or reproducing, or it may be non-viable but still capable ofcarrying out biological and/or biochemical processes such asreplication, transcription, translation, etc.

[0192] An agent is said to have been “purified” if its concentration isincreased, and/or the concentration of one or more undesirablecontaminants is decreased, in a composition relative to the compositionfrom which the agent has been purified. Purification thus encompassesenrichment of an agent in a composition and/or isolation of an agenttherefrom.

[0193] A “solid support” is any solid or semisolid composition to whichan agent can be attached or contained within. Common forms of solidsupport include, but are not limited to, plates, tubes, and beads, allof which could be made of glass or another suitable material, e.g.,polystyrene, nylon, cellulose acetate, nitrocellulose, and otherpolymers. Semisolids and gels that minicells are suspended within arealso considered to be solid supports. A solid support can be in the formof a dipstick, flow-through device, or other suitable configuration.

[0194] A “mutation” is a change in the nucleotide sequence of a generelative to the sequence of the “wild-type” gene. Reference wild-typeeubacterial strains are those that have been cultured in vitro byscientists for decades; for example, a wild-type strain of Escherichiacoli iss E. coli K-12. Mutations include, but are not limited to, pointmutations, deletions, insertions and translocations.

[0195] A “trans-acting regulatory domain” is a regulatory part of aprotein that is expressed from a gene that is not adjacent to the siteof regulatory effect. Trans-acting domains can activate or stimulate(transactivate), or limit or block (transrepress) the gene in question.

[0196] A “reporter gene” refers to a gene that is operably linked toexpression sequences, and which expresses a gene product, typically adetectable polypeptide, the production and detection of which is used asa measure of the robustness and/or control of expression.

[0197] A “detectable compound” or “detectable moiety” produces a signalthat can be detected by spectroscopic, photochemical, biochemical,immunochemical, electromagnetic, radiochemical, or chemical means suchas fluorescence, chemifluoresence, or chemiluminescence, or any otherappropriate means. A “radioactive compound” or “radioactive composition”has more than the natural (environmental) amount of one or moreradioisotopes.

[0198] By “displayed” it is meant that a portion of the membrane proteinis present on the surface of a cell or minicell, and is thus in contactwith the external environment of the cell or minicell. The external,displayed portion of a membrane protein is an “extracellular domain” ora “displayed domain.” A membrane protein may have more than onedisplayed domain, and a minicell of the invention may display more thanone membrane protein.

[0199] A “domain” or “protein domain” is a region of a molecule orstructure that shares common physical and/or chemical features.Non-limiting examples of protein domains include hydrophobictransmembrane or peripheral membrane binding regions, globular enzymaticor receptor regions, and/or nucleic acid binding domains.

[0200] A “transmembrane domain” spans a membrane, a “membrane anchoringdomain” is positioned within, but does not traverse, a membrane. An“extracellular” or “displayed” domain is present on the exterior of acell, or minicell, and is thus in contact with the external environmentof the cell or minicell.

[0201] A “eukaryote” is as the term is used in the art. A eukaryote may,by way of non-limiting example, be a fungus, a unicellular eukaryote, aplant or an animal. An animal may be a mammal, such as a rat, a mouse, arabbit, a dog, a cat, a horse, a cow, a pig, a simian or a human.

[0202] A “eukaryotic membrane” is a membrane found in a eukaryote. Aeukaryotic membrane may, by way of non-limiting example, a cytoplasmicmembrane, a nuclear membrane, a nucleolar membrane, a membrane of theendoplasmic reticulum (ER), a membrane of a Golgi body, a membrane of alysosome a membrane of a peroxisome, a caveolar membrane, or an inner orouter membrane of a mitochondrion, chloroplast or plastid.

[0203] The term “endogenous” refers to something that is normally foundin a cell as that cell exists in nature.

[0204] The term “exogenous” refers to something that is not normallyfound in a cell as that cell exists in nature.

[0205] A “gene” comprises (a) nucleotide sequences that either (i) actas a template for a nucleic acid gene product, or (ii) that encode oneor more open reading frames (ORFs); and (b) expression sequencesoperably linked to (1) or (2). When a gene comprises an ORF, it is a“structural gene.”

[0206] By “immunogenic,” it is meant that a compound elicits productionof antibodies or antibody derivatives and, additionally oralternatively, a T-cell mediated response, directed to the compound or aportion thereof. The compound is an “immunogen.”

[0207] A “ligand” is a compound, composition or moiety that is capableof specifically bound by a binding moiety, including without limitation,a receptor and an antibody or antibody derivative.

[0208] A “membrane protein” is a protein found in whole or in part in amembrane. Typically, a membrane protein has (1) at least one membraneanchoring domain, (2) at least one transmembrane domain, or (3) at leastone domain that interacts with a protein having (1) or (2).

[0209] An “ORF” or “open reading frame” is a nucleotide sequence thatencodes an amino acid sequence of a known, predicted or hypotheticalpolypeptide. An ORF is bounded on its 5′ end by a start codon (usuallyATG) and on its 3′ end by a stop codon (i.e., TAA or TGA). An ORFencoding a 10 amino acid sequence comprises 33 nucleotides (3 for eachof 10 amino acids and 3 for a stop codon). ORFs can encode amino acidsequences that comprise from 10, 25, 50, 125, 150, 175, 200, 250, 300,350, 400, 450, 500, 600, 700, 800, 900 or more amino acids

[0210] The terms “Eubacteria” and “prokaryote” are used herein as theseterms are used by those in the art. The terms “eubacterial” and“prokaryotic” encompasses Eubacteria, including both gram-negative andgram-positive bacteria, prokaryotic viruses (e.g., bacteriophage), andobligate intracellular parasites (e.g., Rickettsia, Chlamydia, etc.).

[0211] An “active site” is any portion or region of a molecule requiredfor, or that regulates, an activity of the molecule. In the case of aprotein, an active site can be a binding site for a ligand or asubstrate, an active site of enzyme, a site that directs or undergoesconformational change in response to a signal, or a site ofpost-translational modification of a protein.

[0212] In a poroplast, the eubacterial outer membrane (OM) and LPS havebeen removed. In a spheroplast, portions of a disrupted eubacterial OMand/or disrupted cell wall either may remain associated with the innermembrane of the minicell, but the membrane is nonetheless porous becausethe permeability of the disrupted OM has been increased. A membrane isthe to be “disrupted” when the membrane's structure has been treatedwith an agent, or incubated under conditions, that leads to the partialdegradation of the membrane, thereby increasing the permeabilitythereof. In contrast, a membrane that has been “degraded” isessentially, for the applicable intents and purposes, removed. Inpreferred embodiments, irrespective of the condition of the OM and cellwall, the eubacterial inner membrane is not disrupted, and membraneproteins displayed on the inner membrane are accessible to compoundsthat are brought into contact with the minicell, poroplast, spheroplast,protoplast or cellular poroplast, as the case may be.

[0213] Host cells (and/or minicells) harboring an expression constructare components of expression systems.

[0214] An “expression vector” is an artificial nucleic acid moleculeinto which an exogenous ORF encoding a protein, or a template of abioactive nucleic acid can be inserted in such a manner so as to beoperably linked to appropriate expression sequences that direct theexpression of the exogenous gene. Preferred expression vectors areepisomal vectors that can replicate independently of chromosomalreplication.

[0215] By the term “operably linked” it is meant that the gene productsencoded by the non-vector nucleic acid sequences are produced from anexpression element in vivo.

[0216] The term “gene product” refers to either a nucleic acid (theproduct of transcription, reverse transcription, or replication) or apolypeptide (the product of translation) that is produced using thenon-vector nucleic acid sequences as a template.

[0217] An “expression construct” is an expression vector into which anucleotide sequence of interest has been inserted in a manner so as tobe positioned to be operably linked to the expression sequences presentin the expression vector. Preferred expression constructs are episomal.

[0218] An “expression element” is a nucleic acid having nucleotidesequences that are present in an expression construct but not itscognate expression vector. That is, an expression element for apolypeptide is a nucleic acid that comprises an ORF operably linked toappropriate expression sequences. An expression element can be removedfrom its expression construct and placed in other expression vectors orinto chromosomal DNA.

[0219] “Expression sequences” are nucleic acid sequences that bindfactors necessary for the expression of genes that have been insertedinto an expression vector. An example of an expression sequence is apromoter, a sequence that binds RNA polymerase, which is the enzyme thatproduces RNA molecules using DNA as a template. An example of anexpression sequence that is both inducible and repressible isL-arabinose operon (araC). See Schleif R. Regulation of the L-arabinoseoperon of Escherichia coli. Trends Genet. December 2000; 16(12):559-65.

[0220] In the present disclosure, “a nucleic acid” or “the nucleic acid”refers to a specific nucleic acid molecule. In contrast, the term“nucleic acid” refers to any collection of diverse nucleic acidmolecules, and thus signifies that any number of different types ofnucleic acids are present. By way of non-limiting example, a nucleicacid may be a DNA, a dsRNA, a tRNA (including a rare codon usage tRNA),an mRNA, a ribosomal RNA (rRNA), a peptide nucleic acid (PNA), a DNA:RNAhybrid, an antisense oligonucleotide, a ribozyme, or an aptamer.

DETAILED DESCRIPTION OF THE INVENTION

[0221] The invention described herein is drawn to compositions andmethods for the production of achromosomal archeabacterial, eubacterialand anucleate eukaryotic cells that are used for diagnostic andtherapeutic applications, for drug discovery, and as research tools.

[0222] The general advantage of minicells over cell-based expressionsystems (e.g., eucaryotic cells or bacterial expression systems) is thatone may express heterologous membrane bound proteins or over expressendogenous membrane bound proteins, cytoplasmic or secreted solubleproteins,or small molecules on the cytoplasmic or extracellular surfacesof the minicells that would otherwise be toxic to live cells. Minicellsare also advantageous for proteins that require a particular lipidenvironment for proper functioning because it is very manipulatable innature. Other advantages include the stability of the minicells due tothe lack of toxicity, the high level of expression that can be achievedin the minicell, and the efficient flexible nature of the minicellexpression system. Such minicells could be used for in vivo targeting orfor selective absorption (i.e., molecular “sponges”) and that thesemolecules can be expressed and “displayed” at high levels. Minicells canalso be used to display proteins for low, medium, high, and ultra highthroughput screening, crystal formation for structure determination, andfor in vitro research use only applications such as transfection.Minicells expressing proteins or small molecules, radioisotopes,image-enhancing reagents can be used for in vivo diagnostics and for invitro diagnostic and assay platforms. Also, soluble and/or membraneassociated signaling cascade elements may be reconstituted in minicellsproducing encapsulated divices to follow extracellular stimulationevents using cytoplasmic reporter events, e.g. transactivation resultingfrom dimerization of dimerization dependant transcriptional activationor repression of said reporter.

[0223] Regarding protein expression, minicells can be engineered toexpress one or more recombinant proteins in order to produce moreprotein per surface area of the particle (at least 10× more protein perunit surface area of protein). The proteins or small molecules that are“displayed” on the minicell surfaces can have therapeutic, discovery ordiagnostic benefit either when injected into a patient or used in aselective absorption mode during dialysis. In vitro assays include drugscreening and discovery, structural proteomics, and other functionalproteomics applications. Proteins that are normally soluble can betethered to membrane anchoring domains or membrane proteins can beexpressed for the purpose of displaying these proteins on the surfacesof the minicell particle in therapeutic, discovery, and diagnosticmodes. The types of proteins that can be displayed include but are notlimited to receptors (e.g., GPCRs, sphingolipid receptors,neurotransmitter receptors, sensory receptors, growth factor receptors,hormone receptors, chemokine receptors, cytokine receptors,immunological receptors, and complement receptors, FC receptors),channels (e.g., potassium channels, sodium channels, calcium channels.),pores (e.g., nuclear pore proteins, water channels), ion and other pumps(e.g., calcium pumps, proton pumps), exchangers (e.g., sodium/potassiumexchangers, sodium/hydrogen exchangers, potassium/hydrogen exchangers),electron transport proteins (e.g., cytochrome oxidase), enzymes andkinases (e.g., protein kinases, ATPases, GTPases, phosphatases,proteases.), structural/linker proteins (e.g., Caveolins, clathrin),adapter proteins (e.g., TRAD, TRAP, FAN), chemotactic/adhesion proteins(e.g., ICAM11, selectins, CD34, VCAM-1, LFA-1, VLA-1), andchimeric/fusion proteins (e.g., proteins in which a normally solubleprotein is attached to a transmembrane region of another protein). As anon-limiting example, the small molecules that can be tethered anddisplayed on the surfaces of the minicells can be carbohydrates (e.g.,monosaccharides), bioactive lipids (e.g., lysosphingolipids, PAF,lysophospholipids), drugs (e.g., antibiotics, ion channelactivators/inhibitors, ligands for receptors and/or enzymes), nucleicacids (e.g., synthetic oligonucleotides), fluorophores, metals, orinorganic and organic small molecules typically found in combinatorialchemistry libraries. Minicells may either contain (encapsulate) ordisplay on their surfaces radionuclides or image-enhancing reagents bothof which could be used for therapeutic and/or diagnostic benefit in vivoor for in vitro assays and diagnostic platforms.

[0224] For in vivo therapeutic uses, minicells can express proteinsand/or display small molecules on their surfaces that would eitherpromote an immune response and passage through the RES system (e.g., toeliminate the minicell and its target quickly), or to evade the RES(e.g., to increase the bioavailability of the minicell). Toxicity isreduced or eliminated because the therapeutic agent is not excreted orprocessed by the liver and thus does not damage the kidneys or liver,because the minicell-based therapeutic is not activated until entry intothe target cell (e.g., in the case of cancer therapeutics or genetherapy). Minicells are of the appropriate size (from about 0.005, 0.1,0.15 or 0.2 micrometers to about 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5micrometers) to facilitate deep penetration into the lungs in the caseswhere administration of the minicell-based therapeutic or diagnostic isvia an inhalant (Strong, A. A., et al. 1987. An aerosol generator systemfor inhalation delivery of pharmacological agents. Med. Instrum.21:189-194). This is due to the fact that minicells can be aerosolized.Without being limited to the following examples, inhalant therapeuticuses of minicells could be applied to the treatment of anaphylacticshock, viral infection, inflammatory reactions, gene therapy for cysticfibrosis, treatment of lung cancers, and fetal distress syndrome.

[0225] Minicells can also display expressed proteins that are enzymesthat may have therapeutic and/or diagnostic uses. The enzymes that aredisplayed may be soluble enzymes that are expressed as fusion proteinswith a transmembrane domain of another protein. Display of such enzymescould be used for in vitro assays or for therapeutic benefit.

[0226] Gene therapy applications afforded by minicells generally involvethe ability of minicells to deliver DNA to target cells (either forreplacement therapy, modifation of cell function or to kill cells).Expression plasmids can be delivered to target cells that would encodeproteins that could be cytoplasmic or could have intracellular signalsequences that would target the protein to a particular organelle (e.g.,mitochondria, nuclei, endoplasmic reticulum, etc.). In the case whereminicells are engulfed by the taget cell, the minicells themselves couldhave these intracellular targeting sequences expressed on their surfacesso that the minicells could be ‘delivered’ to intracellular targets.

[0227] Minicells used for the following therapeutic, discovery, anddiagnostic applications can be prepared as described in this applicationand then stored and/or packaged by a variety of ways, including but notlimited to lyophilization, freezing, mixing with preservatives (e.g.,antioxidants, glycerol), or otherwise stored and packaged in a fashionsimilar to methods used for liposome and proteoliposome formulations.

[0228] The small size of minicells (from about 0.005, 0.1, 0.15 or 0.2micrometers to about 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5 micrometers)makes them suitable for many in vitro diagnostic platforms, includingthe non-limiting examples of lateral flow, ELISA, HTS, especially thoseapplications requiring microspheres or nanospheres that display manytarget proteins or other molecules. The use of protoplast or poroplastminicells may be especially useful in this regard. Assay techniques aredependent on cell or particle size, protein (or molecule to be tested)amount displayed on the surface of the cell or particle, and thesensitivity of the assay being measured. In current whole-cell systems,the expression of the protein of interest is limiting, resulting in thehigher cell number requirement to satisfy the sensitivity of mostassays. However, the relatively large size of cells prevents theincorporation of large numbers of cells in these assays, e.g. 96, 384,and smaller well formats. In contrast, minicells, protoplasts, andporoplasts are smaller in size and can be manipulated to express highlevels of the preselected protein, and can be incorporated into smallwell assay formats.

I. Types of Minicells

[0229] Minicells are derivatives of cells that lack chromosomal DNA andwhich are sometimes referred to as anucleate cells. Because eubacterialand achreabacterial cells, unlike eukaryotic cells, do not have anucleus (a distinct organelle that contains chromosomes), thesenon-eukaryotic minicells are more accurately described as being “withoutchromosomes” or “achromosomal,” as opposed to “anucleate.” Nonetheless,those skilled in the art often use the term “anucleate” when referringto bacterial minicells in addition to other minicells. Accordingly, inthe present disclosure, the term “minicells” encompasses derivatives ofeubacterial cells that lack a chromosome; derivatives of archeabacterialcells that lack their chromosome(s) (Laurence et al., Nucleoid Structureand Partition in Methanococcus jannaschii: An Archaeon With MultipleCopies of the Chromosome, Genetics 152:1315-1323, 1999); and anucleatederivatives of eukaryotic cells. It is understood, however, that some ofthe relevant art may use the terms “anucleate minicells” or anucleatecells” loosely to refer to any of the preceeding types of minicells.

[0230] I.A. Eubacterial Minicells

[0231] One type of minicell is a eubacterial minicell. For reviews ofeubacterial cell cycle and division processes, see Rothfield et al.,Bacterial Cell Division, Annu. Rev. Genet., 33:423-48, 1999; Jacobs etal., Bacterial cell division: A moveable feast, Proc. Natl. Acad. Sci.USA, 96:5891-5893, May, 1999; Koch, The Bacterium's Way for SafeEnlargement and Division, Appl. and Envir. Microb., Vol. 66, No. 9, pp.3657-3663; Bouche and Pichoff, On the birth and fate of bacterialdivision sites. Mol Microbiol, 1998. 29: 19-26; Khachatourians et al.,Cell growth and division in Escherichia coli: a common genetic controlinvolved in cell division and minicell formation. J Bacteriol, 1973.116: 226-229; Cooper, The Escherichia coli cell cycle. Res Microbiol,1990. 141: 17-29; and Danachie and Robinson, “Cell Division: ParameterValues and the Process,” in: Escherichia Coli and SalmonellaTyphimurium: Cellular and Molecular Biology, Neidhardt, Frederick C.,Editor in Chief, American Society for Microbiology, Washington, D.C.,1987, Volume 2, pages 1578-1592, and references cited therein; andLutkenhaus et al., “Cell Division,” Chapter 101 in: Escherichia coli andSalmonella typhimurium: Cellular and Molecular Biology, 2^(nd) Ed.,Neidhardt, Frederick C., Editor in Chief, American Society forMicrobiology, Washington, D.C., 1996, Volume 2, pages 1615-1626, andreferences cited therein. When DNA replication and/or chromosomalpartitioning is altered, membrane-bounded vesicles “pinch off” fromparent cells before transfer of chromosomal DNA is completed. As aresult of this type of dysfunctional division, minicells are producedwhich contain an intact outer membrane, inner membrane, cell wall, andall of the cytoplasm components but do not contain chromosomal DNA. SeeTable 2.

[0232] I.B. Eukaryotic Minicells

[0233] The term “eukaryote” is defined as is used in the art, andincludes any organism classified as Eucarya that are usually classifiedinto four kingdoms: plants, animals, fungi and protists. The first threeof these correspond to phylogenetically coherent groups. However, theeucaryotic protists do not form a group, but rather are comprised ofmany phylogenetically disparate groups (including slime molds, multiplegroups of algae, and many distinct groups of protozoa). See, e.g.,Olsen, G., http://www.bact.wisc.edu/microtextbook/. A type of animal ofparticular interest is a mammal, including, by way of non-limitingexample a rat, a mouse, a rabbit, a dog, a cat, a horse, a cow, a pig, asimian and a human.

[0234] Chromosomeless eukaryotic minicells (i.e., anucleate cells) arewithin the scope of the invention. Platelets are a non-limiting exampleof eukaryotic minicells. Platelets are anucleate cells with little or nocapacity for de novo protein synthesis. The tight regulation of proteinsynthesis in platelets (Smith et al., Platelets and stroke, Vasc Med4:165-72, 1999) may allow for the over-production of exogenous proteinsand, at the same time, under-production of endogenous proteins.Thrombin-activated expression elements such as those that are associatedwith Bcl-3 (Weyrich et al., Signal-dependent translation of a regulatoryprotein, Bcl-3, in activated human platelets, Cel Biology 95:5556-5561,1998) may be used to modulate the expresion of exogneous genes inplatelets.

[0235] As another non-limiting example, eukaryotic minicells aregenerated from tumor cell lines (Gyongyossy-Issa and Khachatourians,Tumour minicells: single, large vesicles released from culturedmastocytoma cells (1985) Tissue Cell 17:801-809; Melton, Cellfusion-induced mouse neuroblastomas HPRT revertants with variant enzymeand elevated HPRT protein levels (1981) Somatic Cell Genet 7:331-344).

[0236] Yeast cells are used to generate fungal minicells. See, e.g., Leeet al., Ibd1p, a possible spindle pole body associated protein,regulates nuclear division and bud separation in Saccharomycescerevisiae, Biochim Biophys Acta 3:239-253, 1999; Kopecka et al., Amethod of isolating anucleated yeast protoplasts unable to synthesizethe glucan fibrillar component of the wall J Gen Microbiol 81:111-120,1974; and Yoo et al., Fission yeast Hrp1, a chromodomain ATPase, isrequired for proper chromosome segregation and its overexpressioninterferes with chromatin condensation, Nucl Acids Res 28:2004-2011,2000. Cell division in yeast is reviewed by Gould and Simanis. Thecontrol of septum formation in fission yeast, Genes & Dev 11:2939-51,1997).

[0237] I.C. Archeabacterial Minicells

[0238] The term “archeabacterium” is defined as is used in the art andincludes extreme thermophiles and other Archaea. Woese, C. R., L.Magrum. G. Fox. 1978. Archeabacteria. Journal of Molecular Evolution.11:245-252. Three types of Archeabacteria are halophiles, thermophilesand methanogens. By physiological definition, the Archaea (informally,archaes) are single-cell extreme thermophiles (includingthermoacidophiles), sulfate reducers, methanogens, and extremehalophiles. The thermophilic members of the Archaea include the mostthermophilic organisms cultivated in the laboratory. The aerobicthermophiles are also acidophilic; they oxidize sulfur in theirenvironment to sulfuric acid. The extreme halophiles are aerobic ormicroaerophilic and include the most salt tolerant organisms known. Thesulfate-reducing Archaea reduce sulfate to sulfide in extremeenvironment. Methanogens are strict anaerobes, yet they gave rise to atleast two separate aerobic groups: the halophiles and athermoacidophilic lineage (Olsen, G.,http://www.bact.wisc.edu/microtextbook/). Non-limiting examples ofhalophiles include Halobacterium cutirubrum and Halogerax mediterranei.Non-limiting examples of methanogens include Methanococcus voltae;Methanococcus vanniela; Methanobacterium thermautotrophicum;Methanococcus voltae; Methanothermusfervidus; and Methanosarcinabarkeri. Non-limiting examples of thermophiles include Azotobactervinelandii; Thermoplasma acidophilum; Pyrococcus horikoshii; Pyrococcusfuriosus; and Crenarchaeota (extremely thermophilic archaebacteria)species such as Sulfolobus solfataricus and Sulfolobus acidocaldarius.

[0239] Archeabacterial minicells are within the scope of the invention.Archeabacteria have homologs of eubacterial minicell genes and proteins,such as the MinD polypeptide from Pyrococcus furiosus (Hayashi et al.,EMBO J 2001 20:1819-28, Structural and functional studies of MinDATPase: implications for the molecular recognition of the bacterial celldivision apparatus). It is thus possible to create Archeabacterialminicells by methods such as, by way of non-limiting example,overexpressing the product of a min gene isolated from a prokaryote oran archeabacterium; or by disrupting expression of a min gene in anarcheabacterium of interest by, e.g., the introduction of mutationsthereof or antisense molecules thereto. See, e.g., Laurence et al.,Nucleoid Structure and Partition in Methanococcus jannaschii: AnArchaeon With Multiple Copies of the Chromosome, Genetics 152:1315-1323,1999.

[0240] In one aspect, the invention is drawn to archael minicells. Byphysiological definition, the Archaea (informally, archaes) aresingle-cell extreme thermophiles (including thermoacidophiles), sulfatereducers, methanogens, and extreme halophiles. The thermophilic membersof the Archaea include the most thermophilic organisms cultivated in thelaboratory. The aerobic thermophiles are also acidophilic; they oxidizesulfur in their environment to sulfuric acid. The extreme halophiles areaerobic or microaerophilic and include the most salt tolerant organismsknown. The sulfate-reducing Archaea reduce sulfate to sulfide in extremeenvironment. Methanogens are strict anaerobes, yet they gave rise to atleast two separate aerobic groups: the halophiles and athermoacidophilic lineage (Olsen, G.,http://www.bact.wisc.edu/microtextbook/).

[0241] I.D. Minicells Produced from Diverse Organisms

[0242] There are genes that can be disrupted to cause minicellproduction that are conserved among the three Kingdoms. For example, SMC(structural maintenance of chormosomes) proteins are conserved amongprokaryotes, archeabacteria and eukaryotes (Hirano, SMC-mediatedchromosome and mechanics: a conserved scheme from bacteria tovertebrates?, Genes and Dev. 13:11-19, 1999; Holmes et al., Closing thering: Links between SMC proteins and chromosome partitioning,condensation, and supercoiling, PNAS 97:1322-1324, 2000; Michiko andHiranol, EMBO J 17:7139-7148, 1998, ATP-dependent aggregation ofsingle-stranded DNA by a bacterial SMC homodimer, 1998). Mutations in B.subtlis smc genes result in the production of minicells (Britton et al.,Characterization of a eubacterial smc protein involved in chromosomepartitioning, Genes and Dev. 12:1254-1259, 1998; Moriya et al., ABacillus subtilis gene-encoding protein homologous to eukaryotic SMCmotor protein is necessary for chromosome partition Mol Microbiol29:179-87, 1998). Disruption of smc genes in various cells is predictedto result in minicell production therefrom.

[0243] As another example, mutations in the yeast genes encoding TRFtopoisomerases result in the production of minicells, and a humanhomolog of yeast TRF genes has been stated to exist (Castano et al., Anovel family of TRF (DNA topoisomerase I-related function) genesrequired for proper nuclear segregation, Nucleic Acids Res 24:2404-10,1996). Mutations in a yeast chromodomain ATPase, Hrp1, result inabnormal chromosomal segregation; (Yoo et al., “Fission yeast Hrp1, achromogomain ATPase, is required for proper chromosome segregation andits overexpression interferes with chromatin condensation,” Nuc. AcidsRes. 28:2004-2001). Disruption of TRF and/or Hrp1 function is predictedto cause minicell production in various cells. Genes involved in septumformation in fission yeast (see, e.g., Gould et al., “The control ofseptum formation in fission yeast,” Genes and Dev. 11:2939-2951, 1997)can be used in like fashion.

[0244] As another example, mutations in the divIVA gene of Bacillussubtilis results in minicell production (Table 2). When expressed in E.coli or the yeast Schizosaccharomyces pombe, a B. subtilis DivIVA-GFPprotein is targeted to cell division sites therein, even though clearhomologs of DivIVA do not seem to exist in E. coli or S. pombe (David etal., Promiscuous targeting of Bacillus subtilis cell division proteinDivIVA to division sites in Escherichia coli and fission yeast, EMBO J19:2719-2727, 2000.) Over- or under-expression of B. subtilis DivIVA ora homolog thereof may be used to reduce minicell production in a varietyof cells.

II. Production of Monicells

[0245] Eubacterial minicells are produced by parent cells having amutation in, and/or overexpressing, or under expressing a gene involvedin cell division and/or chromosomal partitioning, or from parent cellsthat have been exposed to certain conditions, that result in abberantfission of bacterial cells and/or partitioning in abnormal chromosomalsegregation during cellular fission (division). The term “parent cells”or “parental cells” refers to the cells from which minicells areproduced. Minicells, most of which lack chromosomal DNA (Mulder et al.,The Escherichia coli minb mutation resembles gyrB in Defective nucleoidsegregation and decreased negative supercoiling of plasmids. Mol GenGenet, 1990, 221: 87-93), are generally, but need not be, smaller thantheir parent cells. Typically, minicells produced from E. coli cells aregenerally spherical in shape and are about 0.1 to about 0.3 um indiameter, whereas whole E. coli cells are about from about 1 to about 3um in diameter and from about about 2 to about 10 um in length.Micrographs of E. coli cells and minicells that have been stained withDAPI (4:6-diamidino-z-phenylindole), a compound that binds to DNA, showthat the minicells do not stain while the parent E. coli are brightlystained. Such micrographs demonstrate the lack of chromosomal DNA inminicells. (Mulder et al., Mol. Gen. Genet. 221:87-93, 1990).

[0246] As shown in Table 2, minicells are produced by several differentmechanisms such as, by way of non-limiting example, the over expressionof genes involved in chromosomal replication and partitioning, mutationsin such genes, and exposure to various environmental conditions.“Overexpression” refers to the expression of a polypeptide or proteinencoded by a DNA introduced into a host cell, wherein the polypeptide orprotein is either not normally present in the host cell, or wherein thepolypeptide or protein is present in the host cell at a higher levelthan that normally expressed from the endogenous gene encoding thepolypeptide or protein. For example, in E. coli cells that overexpressthe gene product FtsZ (The FtsZ gene encodes a protein that is involvedin regulation of divisions; see Cook and Rothfield, Early stages indevelopment of the Escherichia coli cell-division site. Mol Microbiol,1994. 14: p. 485-495; and Lutkenhaus, Regulation of cell division in E.coli. Trends Genet, 1990. 6: p. 22-25), there is an increase in theformation of minicells (Begg et al., Roles of FtsA and FtsZ in theactivation of division sites. J. Bacteriology, 1997. 180: 881-884).Minicells are also produced by E. coli cells having a mutation in one ormore genes of the min locus, which is a group of genes that encodeproteins that are involved in cell division (de Boer et al., Centralrole for the Escherichia coli minC gene product in two different celldivision-inhibition systems. Proc. Natl. Acad. Sci. USA, 1990. 87:1129-33; Akerlund et al., Cell division in Escherichia coli minBmutants. Mol Microbiol, 1992. 6: 2073-2083).

[0247] Prokaryotes that have been shown to produce minicells includespecies of Escherichia, Shigella, Bacillus, Lactobacillus, andCampylobacter. Bacterial minicell-producing species of particularinterest are E. coli and Bacillus subtilis. E. coli is amenable tomanipulation by a variety of molecular genetic methods, with a varietyof well-characterized expression systems, including many episomalexpression systems, factors and elements useful in the presentinvention. B. subtilis, also amenable to genetic manipulation usingepisomal expression elements, is an important industrial organisminvolved in the production of many of the world's industrial enzymes(proteases, amylases, etc.), which it efficiently produces and secretes.

[0248] In the case of other eubacterial species, homologs of E. coli orB. subilis genes that cause minicell production therein are known or canbe identified and characterized as is known in the art. For example, themin regions of the chromosome of Strepococcus pneumoniae and Neisseriagonorrhoeae have been characterized (Massidda et al., Unconventionalorganization of the division and cell wall gene cluster of Streptococcuspneumoniae, Microbiology 144:3069-78, 1998; and Ramirez-Arcos et al.,Microbiology 147:225-237, 2001 and Szeto et al., Journal of Bacteria183(21):6253, 2001, respectively). Those skilled in the art are able toisolate minicell producing (min) mutants, or prepare compoundsinhibitory to genes that induce a minicell production (e.g., antisenseto min transcripts). TABLE 2 Eubacterial Strains, Mutations andConditions that Promote Minicell Formation Species Strain NotesReferences Campylobacter jejuni may occur naturally late Brock et al.,1987 in growth cycle Bacillus subtilis Mutations in divIVB locus Baraket al., 1999 (inc. minC, minD ripX mutations Sciochetti et al., 1999;Lemon et al., 2001 smc mutations Moriya et al., 1998; Britton et al.,1998 oriC deletions Moriya et al., 1997; Hassan et al., 1997 prfAmutations Pederson and Setlow, 2001 Mutations in divIVA. locus Cha etal., 1997 B.s. 168 ts initiation mutation TsB143 Sargent, 1975 Bacilluscereus WSBC Induced by exposure to Maier et al., 1999 10030 long-chainpolyphosphate Shigella flexneri (2a) MC-1 Gemski et al., 1980 S.dysenteriae (1) MC-V Gemski et al., 1980 Lactobacillus spp. Variantminicell-producing Pidoux et al., 1990 strains isolated from grainsNeisseria gonorrhoeae deletion or overepression Ramirez-Arcos et al.,2001; of min homologues Szeto et al., 2001 Escherichia coli MinAmutations Frazer et al., 1975; Cohen et al. 1976 MinB mutations anddeletions Adler et al., 1967; Davie et al., 1984; Schaumberg et al.;1983; Jaffe et al., 1988; Akerlund et al., 1992 CA8000 cya, crpmutations Kumar et al.; 1979 MukA1 mutation Hiraga et al., 1996 MukE,mukF mutations Yamanaka et al., 1996 hns mutation Kaidow et al., 1995DS410 Heighway et al., 1989 χ1972, χ1776 and χ2076 Curtiss, 1980 P678-54Temperature-sensitive cell Adler et al. 1967; division mutations Alienet al., 1972; Hollenberg et al., 1976 Induced by overexpression De Boeret al., 1988 of minB protein Induced by overexpression of Pichoff etal., 1995 minE protein or derivatives Induced by oveproduction of Wardet al., 1985 ftsZ gene Induced by overexpression Wang et al., 1991 ofsdiA gene Induced by overexpression of min Ramirez-Arcos et al., 2001;genes from Neisseria gonorrhoeae Szeto et al., 2001 Induced by exposureto EGTA Wachi et al., 1999 Legionella Pneumophila Induced by exposure toampicillin Elliot et al., 1985

[0249] Citations for Table 2

[0250] Adler et al., Proc. Natl. Acad. Sci. 57:321-326 (1967)

[0251] Akerlund et al., Mol. Microbiol. 6:2073-2083 (1992)

[0252] Allen et al., Biochem. Biophys. Res. Communi. 47:1074-1079 (1972)

[0253] Barak et al., J. Bacteriol. 180:5237-5333 (1998)

[0254] Britton et al., Genes Dev. 12:1254-9 (1998)

[0255] Brock et al., Can. J. Microbiol. 33:465-470 (1987)

[0256] Cha et al., J. Bacteriol. 179:1671-1683 (1997)

[0257] Cohen et al., Genetics 56:550-551 (1967)

[0258] Curtiss, Roy III, U.S. Pat. No. 4,190,495; Issued Feb. 26, 1980

[0259] Davie et al., J. Bacteriol. 170:2106-2112 (1988)

[0260] Elliott et al., J. Med. Microbiol, 19:383-390 (1985)

[0261] Frazer et al., Curr. Top. Immunol. 69:1-84 (1975)

[0262] Gemski et al., Infect. Immun. 30:297-302 (1980)

[0263] Hassan et al., J. Bacteriol. 179:2494-502 (1997)

[0264] Heighway et al., Nucleic Acids Res. 17:6893-6901 (1989)

[0265] Hiraga et al., J. Bacteriol. 177:3589-3592 (1995)

[0266] Hollenberg et al., Gene 1:33-47 (1976)

[0267] Kumar et al., Mol. Gen. Genet. 176:449-450 (1979)

[0268] Lemon et al., Proc. Natl. Acad. Sci. USA 98:212-7 (2001)

[0269] Maier et al., Appl. Environ. Microbiol. 65:3942-3949 (1999)

[0270] Moriya et al., DNA Res 4:115-26 (1997)

[0271] Moriya et al., Mol. Microbiol. 29:179-87 (1998)

[0272] Markiewicz et al., FEMS Microbiol. Lett. 70:119-123 (1992)

[0273] Pederson and Setlow, J. Bacteriol. 182: 1650-8 (2001)

[0274] Pichoff et al., Mol. Microbiol. 18:321-329 (1995)

[0275] Pidoux et al., J. App. Bacteriol. 69:311-320 (1990)

[0276] Ramirez-Arcos et al. Microbiol. 147:225-237 (2001)

[0277] Sargent M. G., J. Bacteriol. 123:1218-1234 (1975)

[0278] Sciochetti et al., J. Bacteriol. 181:6053-62 (1999)

[0279] Schaumberg et al., J. Bacteriol. 153:1063-1065 (1983)

[0280] Szeto et al., Jour. of Bacter. 183 (21):6253 (2001)

[0281] Wachi et al., Biochimie 81:909-913 (1999)

[0282] Wang et al., Cell 42:941-949 (1985)

[0283] Yamanaka et al., Mol. Gen. Genet. 250:241-251 (1996)

[0284] II.A. Optimized Minicell Construction

[0285] Minicells are produced by several different eubacterial strainsand mechanisms including the overexpression of endogenous or exogenousgenes involved in cell division, chromosomal replication andpartitioning, mutations in such genes, and exposure to various chemicaland/or physical conditions. For example, in E. coli cells thatoverexpress the gene product FtsZ (the ftsZ gene encodes a protein thatis involved in regulation of cell division; see Cook and Rothfield,Early stages in development of the Escherichia coli cell-division site.Mol Microbiol, 1994. 14: p. 485-495; and Lutkenhaus, Regulation of celldivision in E. coli. Trends Genet, 1990. 6: p. 22-25), there is anincrease in the formation of minicells (Begg et al., Roles of FtsA andFtsZ in the activation of division sites. J. Bacteriology, 1997. 180:881-884). Minicells are also produced by E. coli cells having a mutationin one or more genes of the min locus, which is a group of genes thatencode proteins that are involved in cell division (de Boer et al.,Central role for the Escherichia coli minC gene product in two differentcell division-inhibition systems. Proc. Natl. Acad. Sci. USA, 1990. 87:1129-33; Akerlund et al., Cell division in Escherichia coli minBmutants. Mol Microbiol, 1992. 6: 2073-2083).

[0286] Eubacterial cells that have been shown to produce minicellsinclude, but are not limited to species of Escherichia, Shigella,Bacillus, Lactobacillus, Legionella and Campylobacter. Bacterialminicell-producing species of particular interest are E. coli andBacillus subtilis. These organisms are amenable to manipulation by avariety of molecular and genetic methods, with a variety ofwell-characterized expression systems, including many episomal andchromosomal expression systems, as well as other factors and elementsuseful in the present invention.

[0287] The following sections describe genes that may be manipulated soas to stimulate the production of minicells. The invention may includeany of these non-limiting examples for the purpose of preparingminicells. Furthermore, these genes and gene products and conditions,may be used in methodologies to identify other gene(s), gene products,biological events, biochemical events, or physiological events thatinduce or promote the production of minicells. These methodologiesinclude, but are not limited to genetic selection, protein, nucleicacid, or combinatorial chemical library screen, one- or two-hybridanalysis, display selection technologies, e.g. phage or yeast display,hybridization approaches, e.g. array technology, and other high- orlow-throughput approaches.

[0288] II.A.1. Homologs

[0289] Homologs of these genes and gene products from other organismsmay also be used. As used herein, a “homolog” is defined is a nucleicacid or protein having a nucleotide sequence or amino acid sequence,respectively, that is “identical,” “essentially identical,”“substantially identical,” “homologous” or “similar” (as describedbelow) to a reference sequence which may, by way of non-limitingexample, be the sequence of an isolated nucleic acid or protein, or aconsensus sequence derived by comparison of two or more related nucleicacids or proteins, or a group of isoforms of a given nucleic acid orprotein. Non-limiting examples of types of isoforms include isoforms ofdiffering molecular weight that result from, e.g., alternate RNAsplicing or proteolytic cleavage; and isoforms having differentpost-translational modifications, such as glycosylation; and the like.

[0290] Two sequences are said to be “identical” if the two sequences,when aligned with each other, are exactly the same with no gaps,substitutions, insertions or deletions.

[0291] Two sequences are said to be “essentially identical” if thefollowing criteria are met. Two amino acid sequences are “essentiallyidentical” if the two sequences, when aligned with each other, areexactly the same with no gaps, insertions or deletions, and thesequences have only conservative amino acid substitutions. Conservativeamino acid substitutions are as described in Table 3. TABLE 3CONSERVATIVE AMINO ACID SUBSTITUTIONS Type of Amino Groups of AminoAcids that Are Conservative Acid Side Chain Substitutions Relative toEach Other Short side chain Glycine, Alanine, Serine, Threonine andMethionine Hydrophobic Leucine, Isoleucine and Valine Polar Glutamineand Asparagine Acidic Glutamic Acid and Aspartic Acid Basic Arginine,Lysine and Histidine Aromatic Phenylalanine, Tryptophan and Tyrosine

[0292] Two nucleotide sequences are “essentially identical” if theyencode the identical or essentially identical amino acid sequence. As isknown in the art, due to the nature of the genetic code, some aminoacids are encoded by several different three base codons, and thesecodons may thus be substituted for each other without altering the aminoacid at that position in an amino acid sequence. In the genetic code,TTA, TTG, CTT, CTC, CTA and CTG encode Leu; AGA, AGG, CGT, CGC, CGA andCGG encode Arg; GCT, GCC, GCA and GCG encode Ala; GGT, GGC, GGA and GGGencode Gly; ACT, ACC, ACA and ACG encode Thr; GTT, GTC, GTA and GTGencode Val; TCT, TCC, TCA and TCG encode Ser; CCT, CCC, CCA and CCGencode Pro; ATA, ATC and ATA encode Ile; GAA and GAG encode Glu; CAA andCAG encode Gln; GAT and GAC encode Asp; AAT and AAC encode Asn; AGT andAGC encode Ser; TAT and TAC encode Tyr; TGT and TGC encode Cys; AAA andAAG encode Lys; CAT and CAC encode His; TTT and TTC encode Phe, TGGencodes Trp; ATG encodes Met; and TGA, TAA and TAG are translation stopcodons.

[0293] Two amino acid sequences are “substantially identical” if, whenaligned, the two sequences are, (i) less than 30%, preferably ≦20%, morepreferably ≦15%, most preferably ≦10%, of the identities of the aminoacid residues vary between the two sequences; (ii) the number of gapsbetween or insertions in, deletions of and/or subsitutions of, is ≦10%,more preferably ≦5%, more preferably ≦3%, most preferably ≦1%, of thenumber of amino acid residues that occur over the length of the shortestof two aligned sequences.

[0294] Two sequences are said to be “homologous” if any of the followingcriteria are met. The term “homolog” includes without limitationorthologs (homologs having genetic similarity as the result of sharing acommon ancestor and encoding proteins that have the same function indifferent species) and paralog (similar to orthologs, yet gene andprotein similarity is the result of a gene duplication).

[0295] One indication that nucleotide sequences are homologous is if twonucleic acid molecules hybridize to each other under stringentconditions. Stringent conditions are sequence dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength and pH. The Tm isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration isabout 0.02 M at pH 7 and the temperature is at least about 60° C.

[0296] Another way by which it can be determined if two sequences arehomologous is by using an appropriate algorithm to determine if theabove-described criteria for substantially identical sequences are met.Sequence comparisons between two (or more) polynucleotides orpolypeptides are typically performed by algorithms such as, for example,the local homology algorithm of Smith and Waterman (Adv. Appl. Math.2:482, 1981); by the homology alignment algorithm of Needleman andWunsch (J. Mol. Biol. 48:443, 1970); by the search for similarity methodof Pearson and Lipman (Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988); andby computerized implementations of these algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, version10.2 Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.);BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215:403-410,1990); or by visual inspection.

[0297] Optimal alignments are found by inserting gaps to maximize thenumber of matches using the local homology algorithm of Smith andWaterman (1981) Adv. Appl. Math. 2:482-489. “Gap” uses the algorithm ofNeedleman and Wunsch (1970 J Mol. Biol. 48:443-453) to find thealignment of two complete sequences that maximizes the number of matchesand minimizes the number of gaps. In such algorithms, a “penalty” ofabout 3.0 to about 20 for each gap, and no penalty for end gaps, isused.

[0298] Homologous proteins also include members of clusters oforthologous groups of proteins (COGs), which are generated byphylogenetic classification of proteins encoded in complete genomes. Todate, COGs have been delineated by comparing protein sequences encodedin 43 complete genomes, representing 30 major phylogenetic lineages.Each COG consists of individual proteins or groups of paralogs from atleast 3 lineages and thus corresponds to an ancient conserved domain(see Tatusov et al., A genomic perspective on protein families. Science,278: 631-637, 1997; Tatusov et al., The COG database: new developmentsin phylogenetic classification of proteins from complete genomes,Nucleic Acids Res. 29:22-28, 2001; Chervitz et al., Comparisn of theComplete Sets of Worm and Yeast: Orthology and Divergence, Science282:2022-2028, 1998; and http://www.ncbi.nlm.nih.gov/COG/).

[0299] The entirety of two sequences may be identical, essentiallyidentical, substantially identical, or homologous to one another, orportions of such sequences may be identical or substantially identicalwith sequences of similar length in other sequences. In either case,such sequences are similar to each other. Typically, stretches ofidentical or essentially within similar sequences have a length of ≧12,preferably ≧24, more preferably ≧48, and most preferably ≧96 residues.

[0300] II.A.2. Escherichia coli Genes

[0301] Exemplary genes and gene products from E. coli the expressionand/or sequence of which can be manipulated so as to stimulate minicellproduction in E. coli or any other organism, as can homologs thereoffrom any species, include without limitation, the bolA gene (Aldea, M.,et al. 1988. Identification, cloning, and expression of bola, anftsZ-dependent morphogene of Escherichia coli. J. Bacteriol.170:5196-5176; Aldea, M., et al. 1990. Division genes in Escherichiacoli are expressed coordinately to cell septum requirements by gearboxpromoters. EMBO J. 9:3787-3794); the chpA gene (Masuda, Y., et al. 1993.chpA and chpB, Escherichia coli chromosomal homologs of the pem locusresponsible for stable maintenance of plasmid R100. J. Bacteriol.175:6850-6856); the chpB gene (Masuda, Y., et al. 1993. chpA and chpB,Escherichia coli chromosomal homologs of the pem locus responsible forstable maintenance of plasmid R100. J. Bacteriol. 175:6850-6856); thechpR (chpAI) gene (Masuda, Y., et al. 1993. chpA and chpB, Escherichiacoli chromosomal homologs of the pem locus responsible for stablemaintenance of plasmid R100 J. Bacteriol. 175:6850-6856); the chps(chpBI)gene (Masuda, Y., et al. 1993. chpA and chpB, Escherichia colichromosomal homologs of the pem locus responsible for stable maintenanceof plasmid R100 J. Bacteriol. 175:6850-6856); the crg gene (Redfield, R.J., and A. M. Campbell. 1987. Structurae of cryptic lambda prophages. J.Mol. Biol. 198:393-404); the crp gene (Kumar, S., et al. 1979. Controlof minicell producing cell division by cAMP-receptor protein complex inEscherichia coli. Mol. Gen. Genet. 176:449-450); the cya gene (Kumar,S., et al. 1979. Control of minicell producing cell division bycAMP-receptor protein complex in Escherichia coli. Mol. Gen. Genet.176:449-450); the dicA gene (Labie, C., et al. 1989. Isolation andmapping of Escherichia coli mutations conferring resistance to divisioninhibition protein DicB. J. Bacteriol. 171:4315-4319); the dicB gene(Labie, C., et al. 1989. Isolation and mapping of Escherichia colimutations conferring resistance to division inhibition protein DicB. J.Bacteriol. 171:4315-4319; Labie, C., et al. 1990. Minicell-formingmutants of Escherichia coli: suppression of both DicB- andMinD-dependent division inhibition by inactivation of the minC geneproduct. J. Bacteriol. 1990. 172:5852-5858); the dicC gene (Bejar, S.,et al. 1988. Cell division inhibition gene dicB is regulated by a locussimilar to lambdoid bacteriophage immunity loci. Mol. Gen. Genet.212:11-19); the dicF gene (Tetart, F., and J. P. Bouche. 1992.Regulation of the expression of the cell-cycle gene ftsZ by DicFantisense RNA. Division does not require a fixed number of FtsZmolecules. Mol. Microbiol. 6:615-620); the dif gene (Kuempel, P. L., etal. 1991 dif, a recA-independent recombination site in the terminusregion of the chromosome of Escherichia coli. New Biol. 3:799-811); thedksA gene (Yamanaka, K., et al. 1994. Cloning, sequencing, andcharacterization of multicopy suppressors of a mukB mutation inEscherichia coli. Mol. Microbiol. 13:301-312); the dnaK gene (Paek, K.H., and G. C. Walker. 1987. Escherichia coli dnaK null mutants areinviable at high temperature. J. Bacteriol. 169:283-290); the dnaj gene(Hoffman, H. J., et al. 1992. Activity of the Hsp70 chaperonecomplex—DnaK, DnaJ, and GrpE—in initiating phage lambda DNA replicationby sequestering and releasing lambda P protein. Proc. Natl. Acad. Sci.89:12108-12111); the fcsA gene (Kudo, T., et al. 1977. Characteristicsof a cold-sensitive cell division mutant Escherichia coli K-12. Agric.Biol. Chem. 41:97-107); the fic gene (Utsumi, R., et al. 1982.Involvement of cyclic AMP and its receptor protein in filamentation ofan Escherichia coli fic mutant. J. Bacteriol. 151:807-812; Komano, T.,et al. 1991. Functional analysis of the fic gene involved in regulationof cell division. Res. Microbiol. 142:269-277); the fis gene(Spaeny-Dekking, L. et al. 1995. Effects of N-terminal deletions of theEscherichia coli protein Fis on the growth rate, tRNA (2Ser) expressionand cell morphology. Mol. Gen. Genet. 246:259-265); the ftsA gene (Bi,E., and J. Lutkenhaus. 1990. Analysis of ftsZ mutations that conferresistance to the cell division inhibitor SulA (SflA). J. Bacterial.172:5602-5609; Dai, K, and J. Lutkenhaus. 1992. The proper ration ofFtsZ to FtsA is required for cell division to occur in Escherichia coli.J. Bacteriol. 174:6145-6151); the ftsE gene (Taschner, P. E. et al.1988. Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA,pbpB, and ftsE cell division mutants of Escherichia coli duringtemperature shift experiments. J. Bacteriol. 170:1533-1540); the ftsHgene (Ogura, T. et al. 1991. Structure and function of the ftsH gene inEscherichia coli. Res. Microbiol. 142:279-282); the ftsl gene (Begg, K.J., and W. D. Donachie. 1985. Cell shape and division in Escherichiacoli: experiments with shape and division mutants. J. Bacteriol.163:615-622); the ftsJ gene (Ogura, T. et al. 1991. Structure andfunction of the ftsH gene in Escherichia coli Res. Microbiol.142:279-282); the ftsL gene (Guzman, et al. 1992. FtsL, an essentialcytoplasmic membrane protein involved in cell division in Escherichiacoli. J. Bacteriol. 174:7716-7728); the ftsN gene (Dai, K. et al. 1993.Cloning and characterization of ftsN, an essential cell division gene inEscherichia coli isolated as a multicopy suppressor of ftsA12(Ts). J.Bacteriol. 175:3790-3797); the ftsQ gene (Wang, X. D. et al. 1991. Afactor that positively regulates cell division by activatingtranscription of the major cluster of essential cell division genes ofEscherichia coli EMBO J. 10:3362-3372); the ftsW gene (Khattar, M. M. etal. 1994. Identification of FtsW and characterization of a new ftsWdivision mutant of Escherichia coli J. Bacteriol. 176:7140-7147); theftsX (ftsS) gene (Salmond, G. P. and S. Plakidou. 1984. Genetic analysisof essential genes in the ftsE region of the Escherichia coli geneticmap and identification of a new cell division gene, ftsS. Mol. Gen.Genet. 197:304-308); the ftsY gene (Gill, D. R. and G. P. Salmond. 1990.The identification of the Escherichia coli ftsY gene product: an unusualprotein.

[0302] Mol. Microbiol. 4:575-583); the ftsZ gene (Ward, J. E., and J.Lutkenhaus. 1985. Overproduction of FtsZ induces minicell formation.Cell. 42:941-949; Bi, E., and J. Lutkenhaus. 1993. Cell divisioninhibitors SulA and MinCD prevent formation of the FtsZ ring. J.Bacteriol. 175:1118-1125); the gyrB gene (Mulder, E., et al. 1990. TheEscherichia coli minB mutation resembles gyrB in defective nucleoidsegregation and decreased negative supercoiling of plasmids. Mol. Gen.Genet. 221:87-93); the hlfB (ftsH)gene (Herman, C., et al. 1993. Cellgrowth and lambda phage development controlled by the same essentialEscherichia coli gene, ftsH/hflB. Proc. Natl. Acad. Sci.90:10861-10865); the hfq gene (Takada, A., et al. 1999. Negativeregulatory role of the Escherichia coli hfq gene in cell division.Biochem. Biophys. Res. Commun. 266:579-583; the hipA gene (Scherrer, R.,and H. S. Moyed. 1988. Conditional impairment of cell division andaltered lethality in hipA mutants of Escherichia coli K-12. J.Bacteriol. 170:3321-3326); the hipB gene (Hendricks, E. C., et al. 2000Cell division, guillotining of dimer chromosomes and SOS induction inresolution mutants (dif, xerC and xerD) of Escherichia coli Mol.Microbiol. 36:973-981); the hns gene (Kaidow, A., et al. 1995. Anucleatecell production by Escherichia coli delta hns mutant lacking ahistone-like protein, H-NS. J. Bacteriol. 177:3589-3592); the htrB gene(Karow, M., et al. 1991. Complex phenotypes of null mutations in the htrgenes, whole products are essential for Escherichia coli growth atelevated temperatures. Res. Microbiol. 142:289-294); the lpxC (envA)gene(Beall, B., and J. Lutkenhaus. 1987. Sequence analysis, transcriptionalorganization, and insertional mutagenesis of the envA gene ofEscherichia coli J. Bacteriol. 169:5408-5415; Young, K., et al. 1995.The envA permeability/cell division gene of Escherichia coli encodes thesecond enzyme of lipid A biosynthesis.UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase. J. Biol.Chem. 270:30384-30391); the malE gene (Pichoff, S., et al. 1997.MinCD-independent inhibition of cell division by a protein that fusesMalE to the topological specificity factor MinE. J. Bacteriol.179:4616-4619); the minA gene (Davie, E., et al. 1984. Genetic basis ofminicell formation in Escherichia coli K-12. J. Bacteriol.158:1202-1203); the minB gene (Davie, E., et al. 1984. Genetic basis ofminicell formation in Escherichia coli K-12. J. Bacteriol.158:1202-1203); the minC gene (de Boer, P. A., et al. 1990. Central rolefor the Escherichia coli minC gene product in two different celldivision-inhibition systems. Proc. Natl. Acad. Sci. 87:1129-1133); theminD gene (Labie, C., et al. 1990. Minicell-forming mutants ofEscherichia coli: suppression of both DicB- and MinD-dependent divisioninhibition by inactivation of the minC gene product. J. Bacteriol.172:5852-5855; Hayashi, I., et al. 2001. Structural and functionalstudies of MinD ATPase: implications for the molecular recognition ofthe bacterial cell division apparatus. EMBO J. 20:1819-1828); the minegene (de Boer, P. A., et al. 1989. A division inhibitor and atopological specificity factor coded for by the minicell locus determineproper placement of the division septum in E. coli Cell. 56:641-649);the mreB gene (Doi, M., et al. 1988. Determinations of the DNA sequenceof the mreB gene and of the gene products of the mre region thatfunction in formation of the rod shape of Escherichia coli cells. J.Bacteriol. 170:4619-4624); the mreC gene (Wachi, M., et al. 1989. Newmre genes mreC and mreD, responsible for formation of the rod shape ofEscherichia coli cells. J. Bacteriol. 171:6511-6516); the mreD gene(Wachi, M., et al. 1989. New mre genes mreC and mreD, responsible forformation of the rod shape of Escherichia coli cells. J. Bacteriol.171:6511-6516); the mukA gene (Hiraga, S., et al. 1989. Chromosomepartitioning in Escherichia coli: novel mutants producing anucleatecells. J. Bacteriol. 171:1496-1505); the mukB gene (Hiraga, S., et al.1991. Mutants defective in chromosome partitioning in E. coli. Res.Microbiol. 142:189-194); the mukE gene (Yamanaka, K., et al. 1996.Identification of two new genes, mukE and mukF, involved in chromosomepartitioning in Escherichia coli Mol. Gen. Genet. 250:241-251; Yamazoe,M., et al. 1999. Complex formation of MukB, MukE and MukF proteinsinvolved in chromosome partitioning in Escherichia coli EMBO J.18:5873-5884); the mukF gene (Yamanaka, K., et al. 1996. Identificationof two new genes, mukE and mukF, involved in chromosome partitioning inEscherichia coli Mol. Gen. Genet. 250:241-251; Yamazoe, M., et al. 1999.Complex formation of MukB, MukE and MukF proteins involved in chromosomepartitioning in Escherichia coli EMBO J. 18:5873-5884); the parC gene(Kato, J., et al. 1988. Gene organization in the region containing a newgene involved in chromosome partition in Escherichia coli J. Bacteriol.170:3967-3977); the parE gene (Roberts, R. C., et al. 1994. The parDEoperon of the broad-host-range plasmid RK2 specifies growth inhibitionassociated with plasmid loss. J. Mol. Biol. 237:35-51); the pbpA gene(Rodriguez, M. C., and M. A. de Pedro. 1990. Initiation of growth inpbpAts and rodAts mutants of Escherichia coli FEMS Microbiol. Lett.60:235-239); the pcnb gene (Makise, M., et al. 1999. Identification of ahigh-copy-number plasmid suppressor of a lethal phenotype caused bymutant,DnaA protein which has decreased intrinsic ATPase activity. Biol.Pharm. Bull. 22:904-909); the parF (plsC in E. coli ) gene product fromSalmonella (Luttinger, A. L., et al. 1991. A cluster of genes thataffects nucleoid segregation in Salmonella typhimurium. New Biol.3:687-697); the rpoS gene (Cam, K., et al. 1995. Sigma S-dependentoverexpression of ftsz in an Escherichia coli K-12 rpoB mutant that isresistant to the division inhibitors DicB and DicF RNA. Mol. Gen. Genet.248:190-194); the rcsB gene (Gervais, F. G., et al. 1992. The rcsB gene,a positive regulator of colanic acid biosynthesis in Escherichia coli,is also an activator of ftsZ expression. J. Bacteriol. 174:3964-3971);the rcsF gene (Gervais, F. G., and G. R. Drapeau. 1992. Identification,cloning, and characterization of rcsF, a new regulator gene forexopolysaccharide synthesis that suppresses the division mutation ftsZ84in Escherichia coli K-12. J. Bacteriol. 174:8016-8022); the rodA gene(Rodriguez, M. C., and M. A. de Pedro. 1990. Initiation of growth inpbpAts and rodAts mutants of Escherichia coli FEMS Microbiol. Lett.60:235-239); the sdiA (sulB, sfiB) gene (Wang, X. D., et al. 1991. Afactor that positively regulates cell division by activatingtranscription of the major cluster of essential cell division genes ofEscherichia coli EMBO J. 10:3363-3372); the sefA (fabZ) gene (Mohan, S.,et al. 1994. An Escherichia coli gene (FabZ) encoding(3R)-hydroxymyristoyl acyl carrier protein dehydrase. Relation to fabAand suppression of mutations in lipid A biosynthesis. J. Biol Chem.269:32896-32903); the sfiC gene (D' Ari, R., and O. Huisman. 1983. Novelmechanism of cell division inhibition associated with the SOS responsein Escherichia coli J. Bacteriol. 156:243-250); the sulA gene (Bi, E.,and J. Lutkenhaus. 1990. Interaction between the min locus and ftsZ. J.Bacteriol. 172:5610-5616; Bi, E., and J. Lutkenhaus. 1993. Cell divisioninhibitors SulA and MinCD prevent formation of the FtsZ ring. J.Bacteriol. 175:1118-1125); the stfz gene (Dewar, S. J., and W. D.Donachie. 1993. Antisense transcription of the ftsZ-ftsA gene junctioninhibits cell division in Escherichia coli J. Bacteriol. 175:7097-7101);the tolC gene (Hiraga, S., et al. 1989. Chromosome partitioning inEscherichia coli: novel mutants producing anucleate cells. J. Bacteriol.171:1496-1505; Hiraga, S., et al. 1991. Mutants defective in chromosomepartitioning in E. coli Res. Microbiol. 142:189-194); and the zipA gene(Hale, C. A., and P. A. de Boer. 1997. Direct binding of FtsZ to ZipA,an essential component of the septal ring structure that mediates celldivision in E. coli Cell. 88:175-185).

[0303] The guanosine 5′-diphosphate 3′diphosphate (ppGpp) or guanosine5′-triphosphate 3′ diphosphate (pppGpp) nucleotides, collectively(p)ppGpp, found in E. coli or in other members of the Eubacteria,Eucarya or Archaea may be employed to produce minicells (Vinella, D., etal. 1993. Penicillin-binding protein 2 inactivation in Escherichia coliresults in cell division inhibition, which is relieved by FtsZoverexpression. J. Bacteriol. 175:6704-6710; Navarro, F., et al.Analysis of the effect of ppGpp on the ftsQAZ operon in Escherichia coliMol. Microbiol. 29:815-823). The levels, or rate of production of(p)ppGpp may be increased or decreased. By way of non-limiting example,increased (p)ppGpp production results from induction of the stringentresponse. The stringent response in E. coli is a physiological responseelicited by a failure of the capacity for tRNA aminoacylation to keep upwith the demands of protein synthesis. This response can be provokedeither by limiting the availability of amino acids or by limiting theability to aminoacylate tRNA even in the presence of abundant cognateamino acids. Many features of the stringent response behave as if theyare mediated by accumulation of (p)ppGpp. The accumulation of (p)ppGppcan also be provoked by nutritional or other stress conditions inaddition to a deficiency of aminoacyl-tRNA. See Cashel et al., “TheStringent Response,” Chapter 92 in: Escherichia coli and Salmonellatyphimurium: Cellular and Molecular Biology, 2nd Ed., Neidhardt,Frederick C., Editor in Chief, American Society for Microbiology,Washington, D.C., 1996, Volume 1, pages 1458-1496, and references citedtherein.

[0304] By way of non-limiting example, factors that may provoke thestringent response include the 1yt gene or gene product (Harkness, R.E., et al. 1992. Genetic mapping of the 1yta and 1ytB loci ofEscherichia coli, which are involved in penicillin tolerance and controlof the stringent response. Can J. Microbiol. 38:975-978), the relA geneor gene product (Vinella, D., and R. D' Ari. 1994. Thermoinduciblefilamentation in Escherichia coli due to an altered RNA polymerase betasubunit is suppressed by high levels of ppGpp. J. Bacteriol.176:96-972), the relB gene or gene product (Christensen, S. K., et al.2001. RelE, a global inhibitor of translation, is activated duringnutritional stress. Proc. Natl. Acad. Sci. 98:14328-14333), the reIC(rplK) gene or gene product (Yang, X., and E. E. Ishiguro. 2001.Involvement of the N Terminus of Ribosomal Protein Lii in Regulation ofthe RelA Protein of Escherichia coli J. Bacteriol. 183:6532-6537), therelX gene or gene product (St. John, A. C., and A. L. Goldberg. 1980.Effects of starvation for potassium and other inorganic ions on proteindegradation and ribonucleic acid synthesis in Escherichia coli J.Bacteriol. 143:1223-1233), the spoT gene or gene product (Vinella, D.,et al. 1996. Mecillinam resistance in Escherichia coli is conferred byloss of a second activity of the AroK protein. J. Bacteriol.178:3818-3828), the gpp gene or gene product (Keasling, J. D., et al.1993. Guanosine pentaphosphate phosphohydrolase of Escherichia coli is along-chain exopolyphosphatase. Proc. Natl. Acad. Sci. 90:7029-7033), thendk gene or gene product (Kim, H. Y., et al. 1998. Alginate, inorganicpolyphosphate, GTP and ppGpp synthesis co-regulated in Pseudomonasaeruginosa: implications for stationary phase survival and synthesis ofRNA/DNA precursors. Mol. Microbiol. 27:717-725), the rpoB gene or geneproduct (Vinella, D., and R. D' Ari. 1994. Thermoinducible filamentationin Escherichia coli due to an altered RNA polymerase beta subunit issuppressed by high levels of ppGpp. J. Bacteriol. 176:96-972), the rpoCgene or gene product (Bartlett, M. S., et al. 1998. RNA polymerasemutants that destabilize RNA polymerase-promoter complexes alterNTP-sensing by rrn PI promoters. J. Mol. Biol. 279:331-345), the rpoDgene or gene product (Hernandez, V. J., and M. Cashel. 1995. Changes inconserved region 3 of Escherichia coli sigma 70 mediate ppGpp-dependentfunctions in vivo. 252:536-549), gInF gene or gene product (Powell, B.S., and D. L. Court. 1998. Control of ftsZ expression, cell division,and glutamine metabolism in Luria-Bertani medium by the alarmone ppGppin Escherichia coli J. Bacteriol. 180:1053-1062), or gInD gene or geneproduct (Powell, B. S., and D. L. Court. 1998. Control of ftsZexpression, cell division, and glutamine metabolism in Luria-Bertanimedium by the alarmone ppGpp in Escherichia coli J. Bacteriol.180:1053-1062). These genes or gene products, and/or expression thereof,may be manipulated to create minicells.

[0305] II.A.3. Bacillus subtilis Genes

[0306] Exemplary genes and gene products from B. subtilis, theexpression and/or sequence of which can be manipulated so as tostimulate minicell production in B. subtilis or any other organism, ascan homologs thereof from any species, include without limitation, thedivI (divD)gene (Van Alstyne, D., and M. I. Simon. 1971. Divisionmutants of Bacillus subtilis: isolation of PBS 1 transduction ofdivision-specific markers. J. Bacteriol. 108:1366-1379); the divIB (dds,ftsQ) gene (Harry, E. J., et al. 1993. Characterization of mutations indivIB of Bacillus subtilis and cellular localization of the DivIBprotein. Mol. Microbiol. 7:611-621; Harry E. J., et al. 1994. Expressionof divIB of Bacillus subtilis during vegetative growth. J. Bacteriol.176:1172-1179); the divIC gene product from B. subtilis or homologues ofthis gene or gene product found in other members of the Eubacteria,Eucarya or Archaea may be employed to produce minicells (Levin, P. A.,and R. Losick. 1994. Characterization of a cell division gene fromBacillus subtilis that is required for vegetative and sporulation septumformation. J. Bacteriol. 176:1451-1459; Katis, V. L., et al. 1997. TheBacillus subtilis division protein DivIC is a highly abundantmembrane-bound protein that localizes to the division site; the divII(divC) gene (Van Alstyne, D., and M. I. Simon. 1971. Division mutationsof Bacillus subtilis: isolation and PBS 1 transduction ofdivision-specific markers. J. Bacteriol. 108:1366-1379); the divIVA(divD) gene (Cha, J.-H., and G. C. Stewart. 1997. The divIVA minicelllocus of Bacillus subtilis J. Bacteriol. 179:1671-1683); the divIVC(divA) gene (Van Alstyne, D., and M. I. Simon. 1971. Division mutationsof Bacillus subtilis: isolation and PBS1 transduction ofdivision-specific markers. J. Bacteriol. 108:1366-1379); the divV (divB)gene (Van Alstyne, D., and M. I. Simon. 1971. Division mutations ofBacillus subtilis: isolation and PBS1 transduction of division-specificmarkers. J. Bacteriol. 108:1366-1379); the erzA (ytwP) gene (Levin, P.A., et al. 1999. Identification and regulation of a negative regulatorof FtsZ ring formation in Bacillus subtilis Proc. Natl. Acad. Sci.96:9642-9647); the ftsA (spolIN) gene (Feucht, A., et al. 2001.Cytological and biochemical characterization of the FtsA cell divisionprotein of Bacillus subtilis. Mol. Microbiol. 40:115-125); the ftsE gene(Yoshida, K., et al. 1994. Cloning and nucleotide sequencing of a 15 kbregion of the Bacillus subtilis genome containing the io1 operon.Microbiology. 140:2289-2298); the ftsH gene (Deuerling. E., et al. 1995.The ftsH gene of Bacillus subtilis is transiently induced after osmoticand temperature upshift. J. Bacteriol. 177:4105-4112; Wehrl, W., et al.2000. The FtsH protein accumulates at the septum of Bacillus subtilisduring cell division and sporulation. J. Bacteriol. 182:3870-3873); theftsK gene (Sciochetti, S. A., et al. 2001. Identification andcharacterization of the dif Site from Bacillus subtilis J. Bacteriol.183:1058-1068); the ftsL (yIID)gene (Daniel, R. A., et al. 1998.Characterization of the essential cell division gene ftsL (yIID) ofBacillus subtilis and its role in the assembly of the divisionapparatus. Mol. Microbiol. 29:593-604); the ftsW gene (Ikeda, M., et al.1989. Structural similarity among Escherichia coli FtsW and RodAproteins and Bacillus subtilis SpoVE protein, which function in celldivision, cell elongation, and spore formation, respectively. J.Bacteriol. 171:6375-6378); the ftsX gene (Reizer, J., et al. 1998. Anovel protein kinase that controls carbon catabolite repression inbacteria. Mol. Microbiol. 27:1157-1169); the ftsZ gene (Beall, B., andJ. Lutkenhaus). FtsZ in Bacillus subtilis is required for vegetativeseptation and for asymmetric septation during sporulation. Genes andDev. 5:447-45); the gcaD gene (Hove-Jensen, B. 1992. Identification oftms-26 as an allele of the gcaD gene, which encodes N-acetylglucosamine1-phosphate uridyltransferase in Bacillus subtilis J. Bacteriol.174:6852-6856); the gid (ylyC) gene (Kunst, F., et al. 1997. Thecomplete genome sequence of the gram-positive bacterium Bacillussubtilis Nature. 390:237-238); the gidA gene (Ogasawara, N., and H.Yoshikawa. 1992. Genes and their organization in the replication originregion of the bacterial chromosome. Mol. Microbiol. 6:629-634;Nakayashiki, T., and H. Inokuchi. 1998. Novel temperature-sensitivemutants of Escherichia coli that are unable to grow in the absence ofwild-type tRNA6Leu. J. Bacteriol. 180:2931-2935); the gidB gene(Ogasawara, N., and H. Yoshikawa. 1992. Genes and their organization inthe replication origin region of the bacterial chromosome. Mol.Microbiol. 6:629-634; Nakayashiki, T., and H. Inokuchi. 1998. Noveltemperature-sensitive mutants of Escherichia coli that are unable togrow in the absence of wild-type tRNA6Leu. J. Bacteriol. 180:2931-2935);the 1ytC (cw1B) gene (Blackman, S. A., et al. 1998. The role ofautolysins during vegetative growth of Bacillus subtilis 168.Microbiology. 144:73-82); the 1ytD (cw1G) gene (Blackman, S. A., et al.1998. The role of autolysins during vegetative growth of Bacillussubtilis 168. Microbiology. 144:73-82); the 1yte (cw1F) gene (Ishikawa,S., et al. 1998. Regulation of a new cell wall hydrolase gene, cw1F,which affects cell separation in Bacillus subtilis J. Bacteriol.180:23549-2555); the 1ytF (cw1e, yhdD) gene (Ohnishi, R., et al. 1999.Peptidoglycan hydrolase 1ytF plays a role in cell separation with Cw1Fduring vegetative growth of Bacillus subtilis J. Bacteriol.181:3178-1384); the maf gene (Butler, Y. X., et al. 1993. Amplificationof the Bacillus subtilis maf gene results in arrested septum formation.J. Bacteriol. 175:3139-3145); the minC gene (Varley, A. W., and G. C.Stewart. 1992. The divIVB region of the Bacillus subtilis chromosomeencodes homologs of Escherichia coli septum placement (minCD) and cellshape (mreBCD) determinants. J. Bacteriol. 174:6729-6742; Barak, I., etal. 1998. MinCD proteins control the septation process duringsporulation of Bacillus subtilis J. Bacteriol. 180:5327-5333); the minDgene (Varley, A. W., and G. C. Stewart. 1992. The divIVB region of theBacillus subtilis chromosome encodes homologs of Escherichia coli septumplacement (minCD) and cell shape (mreBCD) determinants. J. Bacteriol.174:6729-6742; Barak, I., et al. 1998. MinCD proteins control theseptation process during sporulation of Bacillus subtilis J. Bacteriol.180:5327-5333); the pbpB gene (Daniel, R. A., and J. Errington. 2000.Intrinsic instability of the essential cell division protein FtsL ofBacillus subtilis and a role for DivIB protinein FtsL turnover. Mol.Microbiol. 35:278-289); the ponA gene (Pederson, L. B., et al. Septallocalization of penicillin-binding protein 1 in Bacillus subtilis. J.Bacteriol. 181:3201-3211); the prfA gene (Popham, D. L., and P. Sedlow.1995. Cloning, nucleotide sequence, and mutagenesis of the Bacillussubtilis ponA operon, which codes for penicillin-binding protein (PBP) 1and a PBP-related factor. J. Bacteriol. 177:326-335); the rodB gene(Burdett, I. D. 1979. Electron microscope study of the rod-to-coccusshape change in a temperature-sensitive rod-mutant of Bacillus subtilisJ. Bacteriol. 137:1395-1405;Burdett,, I. D. 1980. Quantitative studiesof rod-coccus morphogenesis in a temperature-sensitive rod-mutant ofBacillus subtilis J. Gen. Microbil. 121:93-103); the secA gene (Sadaie,Y., et al. 1991. Sequencing reveals similarity of the wild-type div +gene of Bacillus subtilis to the Escherichia coli secA gene. Gene.98:101-105); the smc gene (Britton, R. A., et al. 1998 Characterizationof a prokaryotic SMC protein involved in chromosome partitioning. GenesDev. 12:1254-1259; Moriya, S., et al. 1998. A Bacillus subtilisgene-encoding protein homologous to eukaryotic SMC motor protein isnecessary for chromosome partition. Mol. Microbiol. 29:179-187; Hirano,M., and T. Hirano. 1998. ATP-dependent aggregation of single-strandedDNA by a bacterial SMC homodimer. EMBO J. 17:7139-7148); the spoIIE gene(Feucht, a., et al. 1996. Bifunctional protein required for asymmetriccell division and cell-specific transcription in Bacillus subtilis GenesDev. 10:794-803; Khvorova, A., et al. 1998. The spoIIE locus is involvedin the SpoOA-dependent switch in the localization of FtsZ rings inBacillus subtilis J. Bacteriol. 180:1256-1260; Lucet, I., et al. 2000.Direct interaction between the cell division protein FtsZ and the celldifferentiation protein SpoIIE. EMBO J. 19:1467-1475); the spoOA gene(Ireton, K., et al. 1994. spoOJ is required for normal chromosomesegregation as well as the initiation of sporulation in Bacillussubtilis J. Bacteriol. 176:5320-5329); the spoIVF gene (Lee, S., and C.W. Price. 1993. The minCD locus of Bacillus subtilis lacks the minEdeterminant that provides topological specificity to cell division. Mol.Microbiol. 7:601-610); the spoOJ gene (Lin, D. C., et el. 1997. Bipolarlocalization of a chromosome partition protein in Bacillus subtilisProc. Natl. Acad. Sci. 94:4721-4726; Yamaichi, Y., and H. Niki. 2000.Active segregation by the Bacillus subtilis partitioning system inEscherichia coli Proc. Natl. Acad. Sci. 97:14656-14661); the smc gene(Moriya, S., et al. 1998. A Bacillus subtilis gene-encoding proteinhomologous to eukaryotic SMC motor protein is necessary for chromosomepartition. Mol. Microbiol. 29:179-187); the ripX gene (ciochetti, S. A.et al. 1999. The ripX locus of Bacillus subtilis encodes a site-specificrecombinase involved in proper chromosome partitioning. J. Bacteriol.181:6053-6062); and the spoIIIE gene (Wu, L. J., and J. Errington. 1994.Bacillus subtilis spoIIIE protein required for DNA segregation duringasymmetric cell division. Science. 264:572-575); the gene correspondingto the B. subtilis mutant alleal ts-31 (Errington, J., and A. D.Richard. Cell division during growth and sporulation. In A. L.Sonenshein, J. A. Hoch., and R. Losick (eds.). Bacillus subtilis and itsclosest relatives: from genes to cells. American Society forMicrobiology, Washington D.C.); the gene corresponding to the B.subtilis mutant alleal ts-526 (Id.); the yacA gene (Kunst, F., et al.1997. The complete genome sequence of the gram-positive bacteriumBacillus subtilis Nature. 390:237-238); the yfhF gene (Kunst, F., et al.1997. The complete genome sequence of the gram-positive bacteriumBacillus subtilis Nature. 390:237-238); the yfhK gene (Kunst, F., et al.1997. The complete genome sequence of the gram-positive bacteriumBacillus subtilis Nature. 390:237-238); the yjoB gene (Kunst, F., et al.1997. The complete genome sequence of the gram-positive bacteriumBacillus subtilis Nature. 390:237-238); and the ywbG gene (Smith, T. J.,et al. 2000. Autolysins of Bacillus subtilis: multiple enzymes withmultiple functions. Microbiology. 146:249-262).

[0307] II.A.3. Saccharomyes cervisiae Genes

[0308] Exemplary genes and gene products from S. cerevisiae theexpression and/or sequence of which can be manipulated so as tostimulate minicell production in any organism, as can homologs thereoffrom any species, include without limitation, the trf gene productfamily (TRF1, TRF2, TRF3, TRF4, and TRF5) from Saccharomyces cerevisiae(Sadoff, B. U., et al. 1995. Isolation of mutants of Saccharomycescerevisiae requiring DNA topoisomerase I. Genetics. 141:465-479;Castano, I. B., et al. 1996. A novel family of TRF (DNA topoisomeraseI-related function) genes required for proper nuclear segregation.Nucleic Acids Res. 2404-2410); the 1BD1 gene product from Saccharomycescerevisiae (Lee, J., et al. 1999. Ibd1p, a possible spindle pole bodyassociated protein, regulates nuclear division and bud separation inSaccharomyces cerevisiae Biochim. Biophys. Acta. 1449:239-253); thep1o1gene product from Saccharomyces cerevisiae (Cullen, C. F., et al. 2000.A new genetic method for isolating functionally interacting genes: highplo1 (+)-dependent mutants and their suppressors define genes in mitoticand septation pathways in fission yeast. Genetics. 155:1541-1534); thecdc7 locus product(s) from Saccharomyces cerevisiae or homologues ofthis found in other members of the Eubacteria, Eucarya or Archaea may beemployed to produce minicells (Biggins, s. et al. 2001. Genes involvedin sister chromatid separation and segregation in the budding yeastSaccharomyces cerevisiae Genetics. 159:453-470); the cdc15 locusproduct(s) from Saccharomyces cerevisiae or homologues of this found inother members of the Eubacteria, Eucarya or Archaea may be employed toproduce minicells (Mah, A. S., et al. 2001. Protein kinase Cdc15activates the Dbf2-Mob1 kinase complex. Proc. Natl. Acad. Sci.98:7325-7330); the cdc11 locus product(s) from Saccharomyces cerevisiaeor homologues of this found in other members of the Eubacteria, Eucaryaor Archaea may be employed to produce minicells (Fares, H., et al. 1996.Identification of a developmentally regulated septin and involvement ofthe septins in spore formation in Saccharomyces cerevisiae J. Cell Biol.132:399-411); the spg1 locus product(s) from Saccharomyces cerevisiae orhomologues of this found in other members of the Eubacteria, Eucarya orArchaea may be employed to produce minicells (Cullen, C. F., et al.2000. A new genetic method for isolating functionally interacting genes:high plo1(+)-dependent mutants and their suppressors define genes inmitotic and septation pathways in fission yeast. Genetics.155:1521-1534); the sid2 locus product(s) from Saccharomyces cerevisiaeor homologues of this found in other members of the Eubacteria, Eucaryaor Archaea may be employed to produce minicells (Cullen, C. F., et al.2000. A new genetic method for isolating functionally interacting genes:high plo1(+)-dependent mutants and their suppressors define genes inmitotic and septation pathways in fission yeast. Genetics.155:1521-1534); the cdc8 gene product from Saccharomyces cerevisiae(Cullen, C. F., et al. 2000. A new genetic method for isolatingfunctionally interacting genes: high plo1( +)-dependent mutants andtheir suppressors define genes in mitotic and septation pathways infission yeast. Genetics. 155:1521-1534); the rho1 gene product fromSaccharomyces cerevisiae (Cullen, C. F., et al. 2000. A new geneticmethod for isolating functionally interacting genes: highplo1(+)-dependent mutants and their suppressors define genes in mitoticand septation pathways in fission yeast. Genetics. 155:1521-1534); thempd1 gene product from Saccharomyces cerevisiae (Cullen, C. F., et al.2000. A new genetic method for isolating functionally interacting genes:high plo1(+)-dependent mutants and their suppressors define genes inmitotic and septation pathways in fission yeast. Genetics.155:1521-1534); the mpd2 gene product from Saccharomyces cerevisiae(Cullen, C. F., et al. 2000. A new genetic method for isolatingfunctionally interacting genes: high plo1(+)-dependent mutants and theirsuppressors define genes in mitotic and septation pathways in fissionyeast. Genetics. 155:1521-1534); the smy2 gene product fromSaccharomyces cerevisiae (Cullen, C. F., et al. 2000. A new geneticmethod for isolating functionally interacting genes: highplo1(+)-dependent mutants and their suppressors define genes in mitoticand septation pathways in fission yeast. Genetics. 155:1521-1534); thecdc16 gene product from Saccharomyces cerevisiae (Heichman, K. A., andJ. M. Roberts. 1996. The yeast CDC16 and CDC27 genes restrict DNAreplication to once per cell cycle. Cell. 85:39-48); the dma1 geneproduct from Saccharomyces cerevisiae (Murone, M., and V. Simanis. 1996.The fission yeast dma1 gene is a component of the spindle assemblycheckpoint, required to prevent septum formation and premature exit frommitosis if spindle function is compromised. EMBO J. 15:6605-6616); theplo1 gene product from Saccharomyces cerevisiae (Cullen, C. F., et al.2000. A new genetic method for isolating functionally interacting genes:high plo1(+)-dependent mutants and their suppressors define genes inmitotic and septation pathways in fission yeast. Genetics.155:1521-1534); the byr3 gene product from Saccharomyces cerevisiae(Cullen, C. F., et al. 2000. A new genetic method for isolatingfunctionally interacting genes: high plo1(+)-dependent mutants and theirsuppressors define genes in mitotic and septation pathways in fissionyeast. Genetics. 155:1521-1534); the byr4 gene product fromSaccharomyces cerevisiae (Cullen, C. F., et al. 2000. A new geneticmethod for isolating functionally interacting genes: highplo1(+)-dependent mutants and their suppressors define genes in mitoticand septation pathways in fission yeast. Genetics. 155:1521-1534); thepds1 gene product from Saccharomyces cerevisiae (Yamamoto, A., et al.1996. Pds1p, an inhibitor of anaphase in budding yeast, plays a criticalrole in the APC and checkpoint pathway(s). J. Cell Biol. 133:99-110);the espl gene product from Saccharomyces cerevisiae (Rao, H., et al.2001. Degradation of a cohesin subunit by the N-end rule pathway isessential for chromosome stability. Nature. 410:955-999); the ycs4 geneproduct from Saccharomyces cerevisiae (Biggins, S., et al. 2001. Genesinvolved in sister chromatid separation and segregation in the buddingyeast Saccharomyces cerevisiae Genetics. 159:453-470); the cse4 geneproduct from Saccharomyces cerevisiae (Stoler, S. et al. 1995. Amutation in CSE4, an essential gene encoding a novelchromatin-associated protein in yeast, causes chromosome nondisjunctionand cell cycle arrest at mitosis. Genes Dev. 9:573-586); the ip11 geneproduct from Saccharomyces cerevisiae (Biggins, S., and A. W. Murray.2001. The budding yeast protein kinase Ip11/Aurora allows the absence oftension to activate the spindle checkpoint. Genes Dev. 15:3118-3129);the smt3 gene product from Saccharomyces cerevisiae (Takahashi, Y., etal. 1999. Smt3, a SUMO-1 homolog, is conjugated to Cdc3, a component ofseptin rings at the mother-bud neck in budding yeast. Biochem. Biophys.Res. Commun. 259:582-587); the prp16 gene product from Saccharomycescerevisiae (Hotz, H. R., and B. Schwer. 1998. Mutational analysis of theyeast DEAH-box splicing factor Prp16. Genetics. 149:807-815); the prp19gene product from Saccharomyces cerevisiae (Chen, C. H., et al. 2001.Identification and characterization of two novel components of thePrp19p-associated complex, Ntc30p and Ntc20p. J. Biol. Chem.276:488-494); the wss1 gene product from Saccharomyces cerevisiae(Biggins, S., et al. 2001. Genes involved in sister chromatid separationand segregation in the budding yeast Saccharomyces cerevisiae Genetics.159:453-470); the histone H4 gene product from Saccharomyces cerevisiae(Smith, M. M., et al. 1996. A novel histone H4 mutant defective innuclear division and mitotic chromosome transmission. Mol. Cell Biol.16:1017-1026); the histone H3 gene product from Saccharomyces cerevisiae(Smith, M. M., et al. 1996. A novel histone H4 mutant defective innuclear division and mitotic chromosome transmission. Mol. Cell Biol.16:1017-1026); the cse4 gene product from Saccharomyces cerevisiae(Stoler, S., et al. 1995. A mutation in CSE4, an essential gene encodinga novel chromatin-associated protein in yeast, causes chromosomenondisjunction and cell cycle arrest at mitosis. Genes Dev. 9:573-586);the spt4 gene product from Saccharomyces cerevisiae (Basrai, M. A., etal. 1996. Faithful chromosome transmission requires Spt4p, a putativeregulator of chromatin structure in Saccharomyces cerevisiae Mol. CellBiol. 16:2838-2847); the spt5 gene product from Saccharomyces cerevisiae(Yamaguchi, Y., et al. 2001. SPT genes: key players in the regulation oftranscription, chromatin structure and other cellular processes. J.Biochem. (Tokyo). 129:185-191); the spt6 gene product from Saccharomycescerevisiae (Clark-Adams, C. D., and F. Winston. 1987. The SPT6 gene isessential for growth and is required for delta-mediated transcription inSaccharomyces cerevisiae Mol. Cell Biol. 7:679-686); the ndc10 geneproduct from Saccharomyces cerevisiae (Chiang, P. W., et al. 1998.Isolation of murine SPT5 homologue: completion of the isolation andcharacterization of human and murine homologues of yeast chromatinstructural protein complex SPT4, SPT5, and SPT6. Genomics. 47:426-428);the ctf13 gene product from Saccharomyces cerevisiae (Doheny et al.,Identification of essential components of the S. cerevisiae kinetochore,Cell 73:761-774, 1993); the spo1 gene product from Saccharomycescerevisiae (Tavormina et al. 1997. Differential requirements for DNAreplication in the activation of mitotic checkpoints in Saccharomycescerevisiae Mol. Cell Biol. 17:3315-3322); the cwp1 gene product fromSaccharomyces cerevisiae (Tevzadze, G. G., et al. 2000. Spo1, aphospholipase B homolog, is required for spindle pole body duplicationduring meiosis in Saccharomyces cerevisiae Chromosoma. 109:72-85); thedhp1 gene product from Schizosaccharomyces pombe (Shobuike, T., et al.2001. The dhp1(+) gene, encoding a putative nuclear 5′→3′exoribonuclease, is required for proper chromosome segregation infission yeast. Nucleic Acids Res. 29:1326-1333); the rat1 gene productfrom Saccharomyces cerevisiae (Shobuike, T., et al. 2001. The dhp1(+)gene, encoding a putative nuclear 5′→3′ exoribonuclease, is required forproper chromosome segregation in fission yeast. Nucleic Acids Res.29:1326-1333); the hsk1 gene product from Saccharomyces cerevisiae(Masai, H., et al. 1995. hsk1+, a Schizosaccharomyces pombe gene relatedto Saccharomyces cerevisiae CDC7, is required for chromosomalreplication. EMBO J. 14:3094-3104); the dfp1 gene product fromSaccharomyces cerevisiae (Takeda, T., et al. 1999. A fission yeast gene,him1(+)/dfp1(+), encoding a regulatory subunit for Hsk1 kinase, playsessential roles in S-phase initiation as well as in S-phase checkpointcontrol and recovery from DNA damage. Mol. Cell Biol. 19:5535-5547); thedbf4 gene product from Saccharomyces cerevisiae (Weinreich, M., and B.Stillman. 1999. Cdc7p-Dbf4p kinase binds to chromatin during S phase andis regulated by both the APC and the RAD53 checkpoint pathway. EMBO J.18:5334-5346); the rad53 gene product from Saccharomyces cerevisiae(Sun, Z., et al. Spk1/Rad53 is regulated by Mec1-dependent proteinphosphorylation in DNA replication and damage checkpoint pathways. GenesDev. 10:395-406); the ibd1 gene product from Saccharomyces cerevisiae(Lee, J., et al. 1999. Ibd1p, a possible spindle pole body associatedprotein, regulates nuclear division and bud separation in Saccharomycescerevisiae Biochim. Biophys. Acta. 1449:239-253); and the hrp1 geneproduct from Saccharomyces cerevisiae (Henry, M., et al. 1996. PotentialRNA binding proteins in Saccharomyces cerevisiae identified assuppressors of temperature-sensitive mutations in NPL3. Genetics.142:103-115).

[0309] II.B. Gene Expression in Minicells

[0310] II.B.1. In General

[0311] In some aspects of the invention, it may be desirable to alterthe expression of a gene and the production of the corresponding geneproduct. As is known in the art, and is used herein, a “gene product”may be a protein (polypeptide) or nucleic acid. Gene products that areproteins include without limitation enzymes, receptors, transcriptionfactors, termination factors, expression factors, DNA-binding proteins,proteins that effect nucleic acid structure, or subunits of any of thepreceding. Gene products that are nucleic acids include, but are notlimited to, ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), antisenseRNAs, nucleases (including but not limited to catalytic RNAs,ribonucleases, and the like).

[0312] Depending on the function of a gene product, and on the type ofapplication of the invention, it may be desirable to increase proteinproduction, decrease protein production, increase protein nucleic acidproduction and/or increase nucleic acid production. Provided herein arenon-limiting examples of genes and gene products that may bemanipulated, individually or in combination, in order to modulate theexpression of gene products to be included into minicells or parentstrains from which minicells are derived. The expression elements somodulated may be chromosomal and/or episomal, and may be expressedconstitutively or in a regulated fashion, i.e., repressible and/orinducible. Furthermore, gene products under the regulation may be eithermonocistronic or polycistronic with other genes or with themselves.

[0313] II.B.2. Protein Production

[0314] By way of non-limiting example, increased protein production mayoccur through increased gene dosage (increased copy number of a givengene under the control of the native or artificial promotor where thisgene may be on a plasmid or in more than one copy on the chromosome),modification of the native regulatory elements, including, but notlimited to the promotor or operator region(s) of DNA, or ribosomalbinding sites on RNA, relevant repressors/silencers, relevantactivators/enhancers, or relevant antisense nucleic acid or nucleic acidanalog, cloning on a plasmid under the control of the native orartificial promotor, and increased or decreased production of native orartificial promotor regulatory element(s) controlling production of thegene or gene product

[0315] By way of non-limiting example, decreased protein production mayoccur through modification of the native regulatory elements, including,but not limited to the promotor or operator region(s) of DNA, orribosomal binding sites on RNA, relevant repressors/silencers, relevantactivators/enhancers, or relevant antisense nucleic acid or nucleic acidanalog, through cloning on a plasmid under the control of the nativeregulatory region containing mutations or an artificial promotor, eitheror both of which resulting in decreased protein production, and throughincreased or decreased production of native or artificial promotorregulatory element(s) controlling production of the gene or geneproduct.

[0316] As used herein with regards to proteins, “intramolecularactivity” refers to the enzymatic function or structure-dependentfunction. By way of non-limiting example, alteration of intramolecularactivity may be accomplished by mutation of the gene, in vivo or invitro chemical modification of the protein, inhibitor molecules againstthe function of the protein, e.g. competitive, non-competitive, oruncompetitive enzymatic inhibitors, inhibitors that preventprotein-protein, protein-nucleic acid, or protein-lipid interactions,e.g. expression or introduction of dominant-negative ordominant-positive protein or other protein fragment(s), carbohydrate(s),fatty acid(s), lipid(s), and nucleic acid(s) that may act directly orallosterically upon the protein, and/or modification of protein,carbohydrate, fatty acid, lipid, or nucleic acid moieties that modifythe gene or gene product to create the functional protein.

[0317] As used herein with regards to proteins, “intermolecularfunction” refers to the effects resulting from an intermolecularinteraction between the protein or nucleic acid and another protein,carbohydrate, fatty acid, lipid, nucleic acid, or other molecule(s) inor on the cell or the action of a product or products resulting fromsuch an interaction. By way of non-limiting example, intermolecular orintramolecular function may be the act or result of intermolecularphosphorylation, biotinylation, methylation, acylation, glycosylation,and/or other signaling event; this function may be the result of aprotein-protein, protein-nucleic acid, or protein-lipid complex, and/orcarrier function, e.g. the capacity to bind, either covalently ornon-covalently small organic or inorganic molecules, protein(s),carbohydrate(s), fatty acid(s), lipid(s), and nucleic acid(s); thisfunction may be to interact with the membrane to recruit other moleculesto this compartment of the cell; this function may be to regulate thetranscription and/or translation of the gene, other protein, or nucleicacid; and this function may be to stimulate the function of anotherprocess that is not yet described or understood.

[0318] II.B.3. Nucleic Acid Production

[0319] By way of non-limiting example, increased nucleic acid productionmay occur through increased gene dosage (increased copy number of agiven gene under the control of the native or artificial promotor wherethis gene may be on a plasmid or in more than one copy on thechromosome), modification of the native regulatory elements, including,but not limited to the promotor or operator region(s) of DNA, orribosomal binding sites on RNA, relevant repressors/silencers, relevantactivators/enhancers, or relevant antisense nucleic acid or nucleic acidanalog, cloning on a plasmid under the control of the native orartificial promotor, and increased or decreased production of native orartificial promotor regulatory element(s) controlling production of thegene or gene product.

[0320] By way of non-limiting example, decreased nucleic acid productionmay occur through modification of the native regulatory elements,including, but not limited to the promotor or operator region(s) of DNA,or ribosomal binding sites on RNA, relevant repressors/silencers,relevant activators/enhancers, or relevant antisense nucleic acid ornucleic acid analog, through cloning on a plasmid under the control ofthe native regulatory region containing mutations or an artificialpromotor, either or both of which resulting in decreased proteinproduction, and through increased or decreased production of native orartificial promotor regulatory element(s) controlling production of thegene or gene product.

[0321] As used herein with regards to nucleic acids, “intramolecularactivity” refers to a structure-dependent function. By way ofnon-limiting example, alteration of intramolecular activity may beaccomplished by mutation of the gene, in vivo or in vitro chemicalmodification of the nucleic acid, inhibitor molecules against thefunction of the nucleic acid, e.g. competitive, non-competitive, oruncompetitive enzymatic inhibitors, inhibitors that preventprotein-nucleic acid interactions, e.g. expression or introduction ofdominant-negative or dominant-positive protein or other nucleic acidfragment(s), or other carbohydrate(s), fatty acid(s), and lipid(s) thatmay act directly or allosterically upon the nucleic acid or nucleicacid-protein complex, and/or modification of nucleic acid moieties thatmodify the gene or gene product to create the functional nucleic acid.

[0322] As used herein with regards to nucleic acids, “intermolecularfunction” refers to the effects resulting from an intermolecularinteraction between the nucleic acid and another nucleic acid, protein,carbohydrate, fatty acid, lipid, or other molecule(s) in or on the cellor the action of a product or products resulting from such aninteraction. By way of non-limiting example, intermolecular function maybe the act or result of intermolecular or intramolecularphosphorylation, biotinylation, methylation, acylation, glycosylation,and/or other signaling event; this function may be the result of aprotein-nucleic acid, and/or carrier function, e.g. the capacity tobind, either covalently or non-covalently small organic or inorganicmolecules, protein(s), carbohydrate(s), fatty acid(s), lipid(s), andother nucleic acid(s); this function may be to interact with themembrane to recruit other molecules to this compartment of the cell;this function may be to regulate the transcription and/or translation ofthe gene, other nucleic acid, or protein; and this function may be tostimulate the function of another process that is not yet described orunderstood.

[0323] II.C. Genes and Gene Products for Regulation of Expression

[0324] As is known in the art, a variety of genes, gene products andexpression elements may be manipulated, individually or in combination,in order to modulate the expression of genes and/or production geneproducts. These include, by way of non-limiting example, RNApolymerases, ribosomes (ribosomal proteins and ribosomal RNAs), transferRNAs (tRNAs), amino transferases, regulatory elements and promoterregions, transportation of inducible and inhibitory compounds,catabolite repression, general deletions and modifications, cytoplasmicredox state, transcriptional terminators, mechanisms for ribosomaltargeting, proteases, chaperones, export apparatus and membranetargeting, and mechanisms for increasing stability and solubility. Eachof these is discussed in more detail in the following sections.

[0325] II.C.1. RNA Polymerases

[0326] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inminicells. By way of non-limiting example, these techniques may includemodification of an endogenous and/or introduction of an exogenous RNApolymerase. A rpo gene, or any other gene that encodes a RNA polymerasesubunit product from E. coli, or homologs of this gene or its geneproduct found in other prokaryotes, eukaryotes, archaebacteria ororganelles (mitochondria, chloroplasts, plastids and the like) may beemployed to increase the efficiency of gene expression and proteinproduction in parent cells prior to minicell formation and/or insegregated minicells.

[0327] The production or activity of a desired gene product may beincreased by increasing the level and/or activity of an RNA polymerasethat transcribes the gene product's cognate gene. The production oractivity of a desired protein gene product may be increased bydecreasing the level and/or activity of an RNA polymerase thattranscribes a gene product that inhibits the production or function ofthe desired gene product.

[0328] As one example, manipulation of the rpoA (phs, sez) gene or geneproduct from E. coli, or homologs of this gene or gene product found inother members of the Prokaryotes, Eukaryotes, Archaebacteria and/ororganelles (e.g., mitochondria, chloroplasts, plastids and the like) maybe employed to increase the efficiency of gene expression and proteinproduction in parent cells prior to minicell formation and/or insegregated minicells. In addition to rpoA, E. coli. genes that encodeRNA polymerase subunits include rpoB (ftsR, groN, nitB, rif, ron, stl,stv, tabD, sdgB, mbrD), rpoC (tabD), rpoD (alt), rpoE, rpoH (fam, hin,htpR), rpoN (glnF, ntrA), rpoS (abrD, dpeB, katF, nur), and rpoZ (spoS).See Berlyn et al., “Linkage Map of Escherichia coli K-12, Edition 9,”Chapter 109 in: Escherichia coli and Salmonella typhimurium: Cellularand Molecular Biology, 2nd Ed., Neidhardt, Frederick C., Editor inChief, American Society for Microbiology, Washington, D.C., 1996, Volume2, pages 1715-1902, and references cited therein; and Sanderson et al.,“Linkage Map of Salmonella typhimurium, Edition VIII” Chapter 110 in:Escherichia coli and Salmonella typhimurium: Cellular and MolecularBiology, 2nd Ed., Neidhardt, Frederick C., Editor in Chief, AmericanSociety for Microbiology, Washington, D.C., 1996, Volume 2, pages1903-1999, and references cited therein.

[0329] Production of a desired gene product may be preferentially orselectively enhanced by the introduction of an exogenous RNA polymerasethat specifically recognizes expression sequences that are operablylinked to the corresponding gene. By way of non-limiting example, theuse of a T7 RNA polymerase to selectively express genes present onexpression elements that segregate into minicells is described herein.

[0330] II.C.2. Ribosomes

[0331] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inminicells. By way of non-limiting example, these techniques may includemodification of endogenous, and/or addition of exogenous, ribosomes orribosomal subunits. The techniques may be employed to increase theefficiency of gene expression and protein production in parent cellsprior to minicell formation and/or in segregated minicells.

[0332] As is known in the art, a ribosome includes both proteins(polypeptides) and RNA (rRNA). Thus, in the case of a gene that encodesa component of a ribosome, the gene product may be a protein or an RNA.For a review, see Noller et al., “Ribosomes,” Chapter 13 in: Escherichiacoli and Salmonella typhimurium: Cellular and Molecular Biology, 2ndEd., Neidhardt, Frederick C., Editor in Chief, American Society forMicrobiology, Washington, D.C., 1996, Volume 1, pages 167-186, andreferences cited therein. For the sake of convenience, both ribosomalproteins and rRNAs are encompassed by the term “ribosomal subunits.”

[0333] The production or activity of a desired protein gene product maybe increased by increasing the level and/or activity of a ribosomalsubunit that causes or enhances the translation of the desired protein.The production or activity of a desired protein gene product may beincreased by decreasing the level and/or activity of a ribosomal subunitthat causes or enhances translation of a protein that has a negativeimpact on the production or activity of the desired protein.

[0334] Exemplary ribosomal genes and gene products that may bemanipulated include without limitation the E. coli genes rimB, rimC,rimD, rimE, rimF (res), rimG, rimH, rimI, rimJ (tcp), rimK, rimL; rplA,rplB, rplC, rplD, rplE, rplF, rplI, rplJ, rpIK, rplL, rplM, rplN, rplO,rplP, rplQ, rplR, rplS, rplT, rplU, rplV, rplW, rplX, rplY; rpsA, rpsB,rpsC, rpsE (eps, spc, spcA), rpsF (sdgH), rpsG, rpsH, rpsI, rpsJ (nusE),rpsK, rpsL (strA), rpsM, rpsN, rpsO, rpsP, rpsQ, rpsR, rpsS, rpsT, rpsU,rpsV; rrfA, rrfB, rrfC, rrfD, rrfE, rrfF (rrfDbeta, rrvD), rrfG, rrfH;rrlA, rrlB, rrlC, rrlD, rrlE, rrlG, rrlH;, rrnA, rrnB (csqE, rrnB1),rrnC (cqsB), rrnD (cqsD), rrnE (rrnD1), rrnG, rrnH; rrsA, rrsB, rrsC,rrsD, rrsE, rrsG, rrsH, and their cognate gene products.

[0335] Homologs of ribosomal genes or gene products found in othermembers of the Prokaryotes, Eukaryotes, Archaebacteria and organelles(including but not limited to mitochondria, chloroplasts, plastids, andthe like) may be employed to increase the efficiency of gene expressionand protein production in parent cells prior to minicell formationand/or segregated minicells. See, for example, Barkan, A. and M.Goldschmidt-Clermont, Participation of nuclear genes in chioroplast geneexpression, (2000) Biochimie 82:559-572; Willhoeft, U., H. Bu, and E.Tannich, Analysis of cDNA Expressed sequence tags from Entamoebahistolytica: Identification of two highly abundant polyadenylatedtranscripts with no overt open reading frames, (March 1999) Protist150:61-70; Emelyanov, V., Evolutionary relationship of Rickettsiae andmitochondria (February 2001) FEBS Letters 501:11-18; and Gray, M., G.Burger and B. Lang, Mitochondrial Evolution (March 1999) Science283:1476-1481. Ribosomal RNA sequences from a multitude of organisms andorganelles are available through the Ribosomal Database Project (Maidaket al., A new version of the RDP (Ribosomal Database Project) (1999)Nucleic Acids Research 27:171-173). An index of ribosomal proteinsclassified by families on the basis of sequence similarities isavailable on-line at http://www.expasy.ch/cgi-bin/lists?ribosomp.txt;see also (Ramakrishnan et al., Ribosomal protein structures: insightsinto the architecture, machinery and evolution of the ribosome, TIBS23:208-212, 1998.

[0336] II.C.3. Transfer RNAs (tRNAs)

[0337] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inminicells. By way of non-limiting example, these techniques may includeutilization and/or modification of endogenous and/or exogenous transferRNAs (tRNAs). Manipulation of the tRNA genes or gene products from E.coli, or homologs of tRNA genes or gene products found in other membersof the Prokaryotes, Eukaryotes, Archaebacteria and organelles (includingbut not limited to mitochondria, chloroplasts, plastids, and the like)may be employed to increase the efficiency of gene expression andprotein production in parent cells prior to minicell formation and/or insegregated minicells.

[0338] Exemplary E. coli tRNA genes include, but are not limited to, thealaT (talA) gene, the alaU (talD) gene, the alaV gene, the alaW (alaW)gene, the alaX (alaW) gene, the argQ (alaV) gene, the argU (dnaY, pin)gene, the alaU (talD) gene, the argV(argV2) gene, the argW gene, theargX gene, the argY (argV) gene, the argZ (argV) gene, the asnT gene,the asnU gene, the asnV gene, the aspT gene, the aspU gene, the cysTgene, the glnU (supB) gene, the glnV (supE) gene, the gln W (supB) gene,the gltT (tgtB) gene, the gltU (tgtC) gene, the gltV (tgtE) gene, thegltW gene, the glyT (sumA) gene, the glyU (sufD, sumA, sumB, supT) gene,the glyV (ins, mutA) gene, the glyW (ins, mutC) gene, the glyX gene, theglyY gene, the hisR (hisT) gene, the ileT gene, the ileU gene, the ilevgene, the ileX gene, the leuP (leuV) gene, the leuQ (leuV) gene, theleuQ (leuV) gene, the leuT gene, the leuU gene, the leuV (leuV) gene,the leuW (feeB) gene, the leuX (supP) gene, the leuZ gene, the lysTgene, the lysV (supN) gene, the lysW gene, the metT (metT) gene, themetU (metT) gene, the metV (metZ ) gene, the metW gene, the metY gene,the pheU (pheR, pheW) gene, the pheV gene, the proK (proV) gene, theproL (proW) gene, the proM (proU) gene, the serT (divE) gene, the serU(ftsM, supD, supH) gene, the serV (supD) gene, the serW gene, the serX(serW) gene, the thrT gene, the thrU gene, the thrV gene, the thrW gene,the trpT (supU) gene, the tyrT (supC) gene, the tyrU (supM) gene, theatyrV (tyrT, tyrT) gene, the valT gene, the valU (valU) gene, the valV(val) gene, the valW (val) gene, the valX gene, and the valX gene(Komine et al., Genomic Organization and Physical Mapping of theTransfer RNA Genes in Escherichia coli K12. J. Mol. Biol. 212:579-598,1990; Berlyn et al., “Linkage Map of Escherichia coli K-12, Edition 9,”Chapter 109 in: Escherichia Coli and Salmonella Typhimurium: Cellularand Molecular Biology, 2′Ed., Neidhardt, Frederick C., Editor in Chief,American Society for Microbiology, Washington, D.C., 1996, Volume 2,pages 1715-1902, and references cited therein; Sanderson et al.,“Linkage Map of Salmonella typhimurium, Edition VIII” Chapter 110, Id.,pages 1903-1999, and references cited therein; and Hershey, “ProteinSynthesis,” Chapter 40 in: Escherichia Coli and Salmonella Typhimurium:Cellular and Molecular Biology, Neidhardt, Frederick C., Editor inChief, American Society for Microbiology, Washington, D.C., 1987, Volume2, pages 613-647, and references cited therein).

[0339] Also included in the modification of transfer RNA molecules arethe transfer RNA processing enzymes. Exemplary E. coli genes encodingtRNA processing enzymes include, but are not limited to the rnd gene(Blouin R T, Zaniewski R, Deutscher MP. Ribonuclease D is not essentialfor the normal growth of Escherichia coli or bacteriophage T4 or for thebiosynthesis of a T4 suppressor tRNA, J Biol Chem. 258:1423-1426, 1983)and the rnpAB genes (Kirsebom L A, Baer M F, Altman S., Differentialeffects of mutations in the protein and RNA moieties of RNase P on theefficiency of suppression by various tRNA suppressors, J Mol Biol.204:879-888, 1988).

[0340] Also included in the modification of transfer RNA molecules aremodifications in endogenous tmRNAs and/or the introduction of exogenoustmRNAs to minicells and/or their parent cells. The tmRNA (a.k.a. 10SRNA) molecules have properties of tRNAs and mRNAs combined in a singlemolecule. Examples of tmRNAs are described in Zwieb et al. (Survey andSummary: Comparative Sequence Analysis of tmRNA, Nucl. Acids Res.27:21063-2071, 1999).

[0341] II.C.4. Aminoacyl Synthetases

[0342] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inminicells. By way of non-limiting example, these techniques may includeutilization and/or modification of endogenous and/or exogenous aminoacylsynthetases and proteins that effect their production and/or activity.Aminoacyl synthetases are involved in “charging” a tRNA molecule, i.e.,attaching a tRNA to its cognate amino acid. (Martinis et al.,Aminoacyl-tRNA Synthetases: General Features and Relationships. Chapter58 in: Escherichia coli and Salmonella typhimurium: Cellular andMolecular Biology, 2nd Ed., Neidhardt, Frederick C., Editor in Chief,American Society for Microbiology, Washington, D.C., 1996, Volume 1,pages 887-901) and references cited therein; (Grunberg-Manago,Regulation of the Expression of Aminoacyl-tRNA Synthetases andTranslation. Chapter 91 in: Escherichia coli and Salmonella typhimurium:Cellular and Molecular Biology, 2nd Ed., Neidhardt, Frederick C., Editorin Chief, American Society for Microbiology, Washington, D.C., 1996,Volume 1, pages 1432-1457), and references cited therein; and (Hershey,“Protein Synthesis,” Chapter 40 in: Escherichia Coli and SalmonellaTyphimurium: Cellular and Molecular Biology, Neidhardt, Frederick C.,Editor in Chief, American Society for Microbiology, Washington, D.C.,1987, Volume 1, pages 613-647), and references cited therein.

[0343] By way of non-limiting example, manipulation of the aat gene orgene product from E. coli, or homologs of this gene or gene productfound in other members of the Prokaryotes, Eukaryotes, Archaebacteriaand/or organelles (e.g., mitochondria, chloroplasts, plastids and thelike) may be employed to increase the efficiency of gene expression andprotein production in parent cells prior to minicell formation and/or insegregated minicells (Bochner, B. R., and Savageau, M. A. 1979.Inhibition of growth by imidazol(on)e propionic acid: evidence in vivofor coordination of histidine catabolism with the catabolism of otheramino acids. Mol. Gen. Genet. 168(1):87-95).

[0344] In addition to aat, other exemplary E. coli genes encodingaminoacyl synthestases include alaS (act, ala-act, lovB) (Buckel et al.,Suppression of temperature-sensitive aminoacyl-tRNA synthetase mutationsby ribosomal mutations: a possible mechanism. Mol. Gen. Genet.149:51-61, 1976); argS (lovB) (Eriani et al., Isolation andcharacterization of the gene coding for Escherichia coli arginyl-tRNAsynthetase. Nucleic Acids Res. 17:5725-36, 1989); asnS (lcs, tss)(Yamamoto et al., Identification of a temperature-sensitiveasparaginyl-transfer ribonucleic acid synthetase mutant of Escherichiacoli. J. Bacteriol. 132:127-31, 1977); aspS (tls) (Eriani et al.,Aspartyl-tRNA synthetase from Escherichia coli: cloning andcharacterisation of the gene, homologies of its translated amino acidsequence with asparaginyl- and lysl-tRNA syntheases. Nucleic Acids Res.18:7109-18, 1990); cysS (Eriani et al., Cysteinyl-tRNA synthetase:determination of the last E. coli aminoacyl-tRNA synthetase primarystructure. Nucleic Acids Res. 19:265-9, 1991); glnS (Yamao et al.,Escherichia coli glutaminyl-tRNA synthetase. I. Isolation and DNAsequence of the glnS gene. J. Biol. Chem. 257:11639-43, 1982); gltE(Lapointe et al., Thermosensitive mutants of Escherichia coli K-12altered in the catalytic Subunit and in a Regulatory factor of theglutamy-transfer ribonucleic acid synthetase. J. Bacteriol. 122:352-8,1975); gltM (Lapointe et al., Thermosensitive mutants of Escherichiacoli K-12 altered in the catalytic Subunit and in a Regulatory factor ofthe glutamy-transfer ribonucleic acid synthetase. J. Bacteriol.122:352-8, 1975); gltX (Lapointe et al., Thermosensitive mutants ofEscherichia coli K-12 altered in the catalytic Subunit and in aRegulatory factor of the glutamy-transfer ribonucleic acid synthetase.J. Bacteriol. 122:352-8, 1975); glyQ (glySa) (Webster et al., Primarystructures of both subunits of Escherichia coli glycyl-tRNA synthetase,J. Biol. Chem. 252:10637-41, 1983); glyS (act, gly, glySB) (Id.); hisS(Parker et al., Mapping hisS, the structural gene for histidyl-transferribonucleic acid synthetase, in Escherichia coli J. Bacteriol.138:264:7, 1979); ileS (Singer et al., Synthesis of the isoleucyl- andvalyl-tRNA synthetases and the isoleucine-valine biosythetic enzymes ina threonine deaminase regulatory mutant of Escherichia coli K-12. J.Mol. Biol. 175:39-55, 1984); leuS (Morgan et al., Regulation ofbiosythesis of aminoacyl-transfer RNA synthestases and of transfer-RNAin Escherichia coli. Arch. Biol. Med. Exp. (Santiago.) 12:415-26, 1979);lysS (herC, asaD) (Clark et al., Roles of the two lysyl-tRNA synthetasesof Escherichia coli: analysis of nucleotide sequences and mutantbehavior. J. Bacteriol. 172:3237-43, 1990); lysU (Clark et al., Roles ofthe two lysyl-tRNA synthetases of Escherichia coli: analysis ofnucleotide sequences and mutant behavior, J. Bacteriol. 172:3237-43,1990); metG (Dardel et al., Molecular cloning and primary structure ofthe Escherichia coli methionyl-tRNA synthetase gene. J. Bacteriol.160:1115-22, 1984); pheS (phe-act) (Elseviers et al., Molecular cloningand regulation of expression of the genes for initiation factor 3 andtwo aminoacyl-tRNA synthetases, J. Bacteriol. 152:357-62, 1982); pheT(Comer et al., Genes for the alpha and beta subunits of thephenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli J.Bacteriol. 127:923-33, 1976); proS (drp) (Bohman et al., Atemperature-sensitive mutant in prolinyl-tRNA ligase of Escherichia coliK-12 Mo. Gen. Genet. 177:603-5, 1980); serS (Hartlein et al., Cloningand characterization of the gene for Escherichia coli seryl-tRNAsynthetase. Nucleic Acids Res. 15:1005-17, 1987); thrS (Frohler et al.,Genetic analysis of mutations causing borrelidin resistance byoverproduction of threonyl-transfer ribonucleic acid synthetase. J.Bacteriol. 143:1135-41, 1980); trpS (Hall et al., Cloning andcharacterization of the gene for Escherichia coli tryptophanyl-transferribonucleic acid synthetase. J. Bacteriol. 148:941-9, 1981); tyrS(Buonocore et al., Properties of tyrosyl transfer ribonucleic acidsynthetase from two tyrS mutants of Escherichia coli K-12. J. Biol.Chem. 247:4843-9, 1972); and valS (Baer et al., Regulation of thebiosynthesis of aminoacyl-transfer ribonucleic acid synthetases and oftransfer ribonucleic acid in Escherichia coli. V. Mutants with increasedlevels of valyl-transfer ribonucleic acid synthetase. J. Bacteriol.139:165-75, 1979).

[0345] II.C.5. Regulatory Elements and Promoter Regions

[0346] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inminicells. By way of non-limiting example, these techniques may includeutilization and/or modification of regulatory elements and promoterregions. Such manipulations may result in increased or decreasedproduction, and/or changes in the intramolecular and intermolecularfunctions, of a protein in a segregated minicell or its parent cellprior to minicell formation; in the latter instance, the protein may beone that is desirably retained in segregated minicells.

[0347] The production or activity of a desired gene product may beincreased by increasing the level and/or activity of a promoter or otherregulatory region that acts to stimulate or enhance the production ofthe desired gene product. The production or activity of a desired geneproduct may be increased by decreasing the level and/or activity of apromoter or other regulatory region that acts to stimulate or enhancethe production of a gene product that acts to reduce or eliminate thelevel and/or activity of the desired gene product.

[0348] II.C.5.a. Escherichia coli

[0349] Regulatory elements, promoters and other expression elements andexpression factors from E. coli include but are not limited to acrR (Ma,D., et al. 1996. The local repressor AcrR plays a modulating role in theregulation of acrAB genes of Escherichia coli by global stress signals.Mol. Microbiol. 19:101-112); ampD (Lindquist, S., et al. 1989.Signalling proteins in enterobacterial AmpC beta-lactamase regulation.Mol. Microbiol. 3:1091-1102; Holtje, J. V., et al. 1994. The negativeregulator of beta-lactamase induction AmpD is aN-acetyl-anhydromuramyl-L-alanine amidase. FEMS Microbiol. Lett.122:159-164); appR (Diaz-Guerra, L., et al. 1989. appR gene productactivates transcription of microcin C7 plasmid genes. J. Bacteriol.171:2906-2908; Touati, E., et al. 1991. Are appR and katF the sameEscherichia coli gene encoding a new sigma transcription initiationfactor? Res. Microbiol. 142:29-36); appY (Atlung, T., et al. 1989.Isolation, characterization, and nucleotide sequence of appY, aregulatory gene for growth-phase-dependent gene expression inEscherichia coli. J. Bacteriol. 171:1683-1691); araC (Casadaban, M. J.,et al. 1976. Regulation of the regulatory gene for the arabinosepathway, araC. J. Mol. Biol. 104:557-566); arcA (luchi, S., and E. C.Lin. 1988. arcA (dye), a global regulatory gene in Escherichia colimediating repression of enzymes in aerobic pathways. Proc. Natl. Acad.Sci. 85:1888-1892; luchi, S., et al. 1989. Differentiation of arcA,arcB, and cpxA mutant phenotypes of Escherichia coli by sex pilusformation and enzyme regulation. J. Bacteriol. 171:2889-2893); argR(xerA, Rarg) (Kelln, R. A., and V. L. Zak. 1978. Arginine reguloncontrol in a Salmonella typhimurium-Escherichia coli hybrid merodiploid.Mol. Gen Genet. 161:333-335; Vogel, R. H., et al. 1978. Evidence fortranslational repression of arginine biosynthetic enzymes in Escherichiacoli: altered regulation in a streptomycin-resistant mutant. Mol. Gen.Genet. 162:157-162); ascG (Hall, B. G., and L. Xu. Nucleotide sequence,function, activation, and evolution of the cryptic asc operon ofEscherichia coli K12. Mol. Biol. Evol. 9:688-706); asiB (Bennik, M. H.,et al. 2000. Defining a rob regulon in Escherichia coli by usingtransposon mutagenesis. J. Bacteriol. 182:3794-3801); asnC (Kolling, R.,and H. Lother. 1985. AsnC: an autogenously regulated activator ofasparagine synthetase A transcription in Escherichia coli. J. Bacteriol.164:310-315); atoC (Jenkins, L. S., and W. D. Nunn. 1987. Regulation ofthe ato operon by the atoC gene in Escherichia coli J. Bacteriol.169:2096-2102); baeR (Nagasawa, S., et al. 1993. Novel members of thetwo-component signal transduction genes in Escherichia coli J. Biochem.(Tokyo). 114:350-357); baeS (Id.Id.); barA (Nagasawa, S., et al. 1992. Anovel sensor-regulator protein that belongs to the homologous family ofsignal-transduction proteins involved in adaptive responses inEscherichia coli Mol. Microbiol. 6:799-807; Ishige, K., et al. 1994. Anovel device of bacterial signal transducers. EMBO J. 13:5195-5202);basS (Nagasawa, S., et al. 1993. Novel members of the two-componentsignal transduction genes in Escherichia coli J. Biochem. (Tokyo).114:350-357); betI (Lamark, T., et al. 1996. The complex bet promotersof Escherichia coli: regulation by oxygen (ArcA), choline (BetI), andosmotic stress. J. Bacteriol. 178:1655-1662); bglG (bglC, bglS)(Schnetz, K., and B. Rak. 1988. Regulation of the bgl operon ofEscherichia coli by transcriptional antitermination. EMBO J.7:3271-3277; Schnetz, K., and B. Rak. 1990. Beta-glucoside permeaserepresses the bgl operon of Escherichia coli by phosphorylation of theantiterminator protein and also interacts with glucose-specific enzymeIII, the key element in catabolite control. Proc. Natl. Acad. Sci.87:5074-5078); birA (bioR, dhbB) (Barker, D. F., and A. M. Campbell.1981. Genetic and biochemical characterization of the birA gene and itsproduct: evidence for a direct role of biotin holoenzyme synthetase inrepression of the biotin operon in Escherichia coli. J. Mol. Biol.146:469-492; Barker, D. F., and A. M. Campbell. 1981. The birA gene ofEscherichia coli encodes a biotin holoenzyme synthetase. J. Mol. Biol.146:451-467; Howard, P. K., et al. 1985. Nucleotide sequence of the birAgene encoding the biotin operon repressor and biotin holoenzymesynthetase functions of Escherichia coli Gene. 35:321-331); btuR(Lundrigan, M. D., et al. 1987. Separate regulatory systems for therepression of mete and btuB by vitamin B12 in Escherichia coli. Mol.Gen. Genet. 206:401-407; Lundrigan, M. D., and R. J. Kadner. 1989.Altered cobalamin metabolism in Escherichia coli btuR mutants affectsbtuB gene regulation. J. Bacteriol. 171:154-161); cadC (Watson, N., etal. 1992. Identification of elements involved in 5 transcriptionalregulation of the Escherichia coli cad operon by external pH. J.Bacteriol. 174:530-540); celD (Parker, L. L., and B. G. Hall. 1990.Characterization and nucleotide sequence of the cryptic cel operon ofEscherichia coli K12. Genetics. 124:455-471); chaB (Berlyn, M. K. B., etal. 1996. Linkage map of Escherichia coli K-12, Edition 9. In F. C.Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B.Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger(eds.). Escherichia coli and Salmonella typhimurium: cellular andmolecular biology, 2nd ed. American Society for Microbiology, WashingtonD.C.); chaC (Berlyn, M. K. B., et al. 1996. Linkage map of Escherichiacoli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E.C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.Schaechter, and H. E. Umbarger (eds.). Escherichia coli and Salmonellatyphimurium: cellular and molecular biology, 2nd ed. American Societyfor Microbiology, Washington D.C.); cpxR (Danese, P. N., et al. 1995.The Cpx two-component signal transduction pathway of Escherichia coliregulates transcription of the gene specifying the stress-inducibleperiplasmic protease, DegP. Genes Dev. 9:387-398); crl (Arnqvist, A., etal. 1992. The Crl protein activates cryptic genes for curli formationand fibronectin binding in Escherichia coli HB101. Mol. Microbiol.6:2443-2452); cspA (Bae, W., et al. 1999 Characterization of Escherichiacoli cspE, whose product negatively regulates transcription of cspA, thegene for the major cold shock protein. Mol. Microbiol. 31:1429-1441);cspE (Id.); csrA (Liu, M. Y., et al. 1995. The product of thepleiotropic Escherichia coli gene csrA modulates glycogen biosynthesisvia effects on mRNA stability. J. Bacteriol. 177:2663-2672); cynR(Anderson, P. M., et al. 1990. The cyanase operon and cyanatemetabolism. FEMS Microbiol. Rev. 7:247-252; Sung, Y. C., and J. A.Fuchs. 1992. The Escherichia coli K-12 cyn operon is positivelyregulated by a member of the lysR family. J. Bacteriol. 174:3645-3650);cysB (Jagura-Burdzy, G., and D. Hulanicka. 1981. Use of gene fusions tostudy expression of cysB, the regulatory gene of the cysteine regulon.J. Bacteriol. 147:744-751); cytR (Hammer-Jespersen, K., and A.Munch-Ptersen. 1975. Multiple regulation of nucleoside catabolizingenzymes: regulation of the deo operon by the cytR and deoR geneproducts. Mol. Gen. Genet. 137:327-335); dadQ (alnR) (Wild, J., and B.Obrepalska. 1982. Regulation of expression of the dadA gene encodingD-amino acid dehydrogenase in Escherichia coli: analysis of dadA-lacfusions and direction of dadA transcription. Mol. Gen. Genet.186:405-410); dadR (alnR) (Wild, J., et al. 1985. Identification of thedadX gene coding for the predominant isozyme of alanine racemase inEscherichia coli K12. Mol. Gen. Genet. 198:315-322); deoR (nucR, tsc,nupG) (Hammer-Jespersen, K., and A. Munch-Ptersen. 1975. Multipleregulation of nucleoside catabolizing enzymes: regulation of the deooperon by the cytR and deoR gene products. Mol. Gen. Genet.137:327-335); dgoR (Berlyn, M. K. B., et al. 1996. Linkage map ofEscherichia coli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L.Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M.Riley, M. Schaechter, and H. E. Umbarger (eds.). Escherichia coli andSalmonella typhimurium: cellular and molecular biology, 2nd ed. AmericanSociety for Microbiology, Washington D.C.); dicA (Bejar, S., et al.1988. Cell division inhibition gene dicB is regulated by a locus similarto lambdoid bacteriophage immunity loci. Mol. Gen. Genet. 212:11-19);dnaK (gro, groP, groPAB, groPC, groPF, grpC, grpF, seg) (Bochner, B. R.,et al. 1986. Escherichia coli DnaK protein possesses a 5′-nucleotidaseactivity that is inhibited by AppppA. J. Bacteriol. 168:931-935); dniR(Kajie, S., et al. 1991. Molecular cloning and DNA sequence of dniR, agene affecting anaerobic expression of the Escherichia coli hexahemenitrite reductase. FEMS Microbiol. Lett. 67:205-211); dsdC (Heincz, M.C., and E. McFall. 1978. Role of the dsdC activator in regulation ofD-serine deaminase synthesis. J. Bacteriol. 136:96-103); ebgR (Hall, B.G., and N. D. Clarke. 1977. Regulation of newly evolved enzymes. IIIEvolution of the ebg repressor during selection for enhanced lactaseactivity. Genetics. 85:193-201); envY (Lundrigan, M. D., and C. F.Earhart. 1984. Gene envY of Escherichia coli K-12 affectsthermoregulation of major porin expression. J. Bacteriol. 157:262-268);envZ (ompB, perA, tpo) (Russo, F. D, and T. J. Silhavy. 1991. EnvZcontrols the concentration of phosphorylated OmpR to mediateosmoregulation of the porin genes. J. Mol. Biol. 222:567-580); evgA(Nishino, K., and A. Yamaguichi. 2001. Overexpression of the responseregulator evgA of the two-component signal transduction system modulatesmultidrug resistance conferred by multidrug resistance transporters. J.Bacteriol. 183:1455-1458); evgS (Id.); exuR (Portalier, R., et al. 1980.Regulation of Escherichia coli K-12 hexuronate system genes: exuregulon. J. Bacteriol. 143:1095-1107); fadR (dec, ole, thdB) (Simons, R.W., et al. 1980. Regulation of fatty acid degradation in Escherichiacoli: isolation and characterization of strains bearing insertion andtemperature-sensitive mutations in gene fadR. J. Bacteriol.142:621-632); fecI (Van Hove, B., et al. 1990. Novel two-componenttransmembrane transcription control: regulation of iron dicitratetransport in Escherichia coli K-12. J. Bacteriol. 172:6749-6758); fecR(Id.); fhlA (Maupin, J. A., and K. T. Shanmugam. 1990. Geneticregulation of formate hydrogenlyase of Escherichia coli: role of thefhlA gene product as a transcriptional activator for a new regulatorygene, fhlB. J. Bacteriol. 172:4798-4806; Rossmann, R., et al. 1991.Mechanism of regulation of the formate-hydrogenlyase pathway by oxygen,nitrate, and pH: definition of the formate regulon. Mol. Microbiol.5:2807-2814); fhlB (Maupin, J. A., and K. T. Shanmugam. 1990. Geneticregulation of formate hydrogenlyase of Escherichia coli: role of thefhlA gene product as a transcriptional activator for a new regulatorygene, fhlB. J. Bacteriol. 172:4798-4806); fimB (pil) (Pallesen, L., etal. 1989. Regulation of the phase switch controlling expression of type1 fimbriae in Escherichia coli Mol. Microbiol. 3:925-931); fimE (pilH)(Id.); flhC (flal) (Liu, X., and P. Matsumura. 1994. The FlhD/FlhCcomplex, a transcriptional activator of the Escherichia coli flagellarclass II operons. J. Bacteriol. 176:7345-7351); flhD (flhB) (Id.); fliA(flaD, rpoF) (Komeda, Y., et al. 1986. Transcriptional control offlagellar genes in Escherichia coli K-12. J. Bacteriol. 168:1315-1318);fnr (frdB, nirA, nirR) (Jones, H. M., and R. P. Gunsalus. 1987.Regulation of Escherichia coli fumarate reductase (frdABCD) operonexpression by respiratory electron acceptors and the fnr gene product.J. Bacteriol. 169:3340-3349); fruR (fruC, shl) (Geerse, R. H., et al.The PEP fructose posphotransferase system in Salmonella typhimurium: FPrcombines enzyme IIIFru and pseudo-HPr activities. Mol. Gen. Genet.216:517-525); fucR (Zhu, Y., and E. C. Lin. 1986. An evolvant ofEscherichia coli that employs the L-fucose pathway also for growth onL-galactose and D-arabinose. J. Mol. Evol. 23:259-266); fur (Bagg, A.,and J. B. Neilands. 1987. Ferric uptake regulation protein acts as arepressor, employing iron (II) as a cofactor to bind the operator of aniron transport operon in Escherichia coli Biochemistry 26:5471-5477);gadR gene product from Lactococcus lactis (Sanders, J. W., et al. 1997.A chloride-inducible gene expression cassette and its use in inducedlysis of Lactococcus lactis. Appl. Environ. Microbiol. 63:4877-4882);galR (von Wilcken-Bergmann, B., and B. Muller-Hill. 1982. Sequence ofgalR gene indicates a common evolutionary origin of lac and galrepressor in Escherichia coli Proc. Natl. Acad. Sci. 79:2427-2431); gals(mglD) (Weickert., M. J., and S. Adhya. 1992. Isorepressor of the galregulon in Escherichia coli. J. Mol. Biol. 226:69-83); galU (Berlyn, M.K. B., et al. 1996. Linkage map of Escherichia coli K-12, Edition 9. InF. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B.Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger(eds.). Escherichia coli and Salmonella typhimurium: cellular andmolecular biology, 2nd ed. American Society for Microbiology, WashingtonD.C.); gatR (Nobelmann, B., and J. W. Lengeler. 1996. Molecular analysisof the gat genes from Escherichia coli and of their roles in galactitoltransport and metabolism. J. Bacteriol. 178:6790-6795); gcvA (Wilson, R.L., et al. 1993. Positive regulation of the Escherichia coli glycinecleavage enzyme system. J. Bacteriol. 175:902-904); glgs (Hengge-Aronis,R., et al. 1993. Osmotic regulation of rpoS-dependent genes inEscherichia coli. J. Bacteriol. 175:259-265; Yang, H., et al. 1996.Coordinate genetic regulation of glycogen catabolism and biosynthesis inEscherichia coli via the CsrA gene product. J. Bactgeriol.178:1012-1017); glnB (Bueno, R., et al. 1985. Role of glnB and glnD geneproducts in regulation of the glnALG operon of Escherichia coli. J.Bacteriol. 164:816-822); glnG (gln, ntrC) (Pahel, G., and B. Tyler.1979. A new glnA-linked regulatory gene for glutamine synthetase inEscherichia coli. Proc. Natl. Acad. Sci. 76:4544-4548); glnL (glnR,ntrB) (MacNeil, T., et al. The products of glnL and glnG arebifunctional regulatory proteins. Mol. Gen. Genet. 188:325-333); glpR(Silhavy, T. J., et al. 1976. Periplasmic protein related to thesn-glycerol-3-phosphate transport system of Escherichia coli. J.Bacteriol. 126:951-958); gltF (Castano, I., et al. gltF, a member of thegltBDF operon of Escherichia coli, is involved in nitrogen-regulatedgene expression. Mol. Microbiol. 6:2733-2741); gntR (Peekhaus, N., andT. Conway. 1998. Positive and negative transcriptional regulation of theEscherichia coli gluconate regulon gene gntT by GntR and the cyclic AMP(cAMP)-cAMP receptor protein complex. J. Bacteriol. 180:1777-1785); hha(Neito, J. M., et al. The hha gene modulates haemolysin expression inEscherichia coli. Mol. Microbiol. 5:1285-1293); himD (hip) (Goosen, N.,et al. 1984. Regulation of Mu transposition. II. The Escherichia coliHimD protein positively controls two repressor promoters and the earlypromoter of bacteriophage Mu. Gene. 32:419-426); hrpB gene product fromPseudomonas solanacearum (Van Gijsegem, F., et al. 1995. The hrp genelocus of Pseudomonas solanacearum, which controls the production of atype III secretion system, encodes eight proteins related to componentsof the bacterial flagellar biogenesis complex. Mol. Microbiol.15:1095-1114); hybF (Berlyn, M. K. B., et al. 1996. Linkage map ofEscherichia coli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L.Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M.Riley, M. Schaechter, and H. E. Umbarger (eds.). Escherichia coli andSalmonella typhimurium: cellular and molecular biology, 2nd ed. AmericanSociety for Microbiology, Washington D.C.); hycA (Hopper, S., et al.1994. Regulated expression in vitro of genes coding for formatehydrogenlyase components of Escherichia coli. J. Biol. Chem.269:19597-19604); hydG (Leonhartsberger, S. et al. 2001. The hydH/Ggenes from Escherichia coli code for a zinc and lead responsivetwo-component regulatory system. J. Mol. Biol. 307:93-105); hydH (Id.);iciA (Thony, B., et al. 1991. iciA, an Escherichia coli gene encoding aspecific inhibitor of chromosomal initiation of replication in vitro.Proc. Natl. Acad. Sci. 88:4066-4070); icIR (Maloy, S. R., and W. D.Nunn. 1982. Genetic regulation of the glyoxylate shunt in Escherichiacoli K-12. J. Bacteriol. 149:173-180); ileR (avr, firA) (Johnson, D. I.,and R. L. Somerville. 1984. New regulatory genes involved in the controlof transcription initiation at the thr and ilv promoters of Escherichiacoli K-12. Mol. Gen. Genet. 195:70-76); ilvR (Id.); ilvU (Fayerman, J.T., et al. 1979. ilvU, a locus in Escherichia coli affecting thederepression of isoleucyl-tRNA synthetase and the RPC-5 chromatographicprofiles of tRNAIle and tRNAVal. J. Bio. Chem. 254:9429-9440); ilvY(Wek, R. C., and G. W. Hatfield. 1988. Transcriptional activation atadjacent operators in the divergent-overlapping ilvY and ilvC promotersof Escherichia coli J. Mol. Biol. 203:643-663); inaA (White, S., et al.1992. pH dependence and gene structure of inaA in Escherichia coli J.Bacteriol. 174:1537-1543); inaR (Id.); kdgR (Nemoz, G., et al. 1976.Physiological and genetic regulation of the aldohexuronate transportsystem in Escherichia coli. J. Bacteriol. 127:706-718); lacI (Riggs, A.D, and S. Bourgeois. 1968. On the assay, isolation and characterizationof the lac repressor. J. Mol. Biol. 34:361-364); leuO (Shi, X., and G.N. Bennett. 1995. Effects of multicopy LeuO on the expression of theacid-inducible lysine decarboxylase gene in Escherichia coli. J.Bacteriol. 177:810-814; Klauck, E., et al. 1997. The LysR-like regulatorLeuO in Escherichia coli is involved in the translational regulation ofrpoS by affecting the expression of the small regulatory DsrA-RNA. Mol.Microbiol. 25:559-569); leur (Theall, G., et al. 1979. Regulation of thebiosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichiacoli. IV. Mutants with increased levels of leucyl- or seryl-tRNAsynthetase. Mol. Gen. Genet. 169:205-211); leuY (Morgan, S., et al.1979. Regulation of biosynthesis of aminoacyl-transfer RNA synthetasesand of transfer-RNA in Escherichia coli Arch. Biol. Med. Exp. (Santiago)12:415-426); lexA (Mount, D. W. 1977. A mutant of Escherichia colishowing constitutive expression of the lysogenic induction anderror-prone DNA repair pathways. Proc. Natl. Acad. Sci. 74:300-304;Little, J. W., et al. 1980. Cleavage of the Escherichia coli lexAprotein by the recA protease. Proc. Natl. Acad. Sci. 77:3225-3229); lldR(lctR) (Dong, J. M., et al. 1993. Three overlapping lct genes involvedin L-lactate utilization by Escherichia coli. J. Bacteriol.175:6671-6678); lpp (Brosius, J. Expression vectors employing lambda-,trp-, lac-, and lpp-derived promoters. 1988. Biotechnology. 10:205-225);lrhA (genR) (Bongaerts, J., et al. 1995. Transcriptional regulation ofthe proton translocating NADH dehydrogenase genes (nuoA-N) ofEscherichia coli by electron acceptors, electron donors and generegulators. Mol. Microbiol. 16:521-534); lrp (ihb, livR, lss, lstR,oppl, rblA, mbf) (Ito, K., et al. Multiple control of Escherichia colilysyl-tRNA synthetase expression involves a transcriptional repressorand a translational enhancer element. Proc. Natl. Acad. Sci.90:302-306); lysR (Gicquel-Sanzey, B. and P. Cossart. 1982. Homologiesbetween different procaryotic DNA-binding regulatory proteins andbetween their sites of action. EMBO J. 1:591-595; Stragier, P., et al.1983. Regulation of diaminopimelate decarboxylase synthesis inEscherichia coli. II. Nucleotide sequence of the lysA gene and itsregulatory region. J. Mol. Biol. 168:321-331); malI (Reidl, J., et al.1989. MalI, a novel protein involved in regulation of the maltose systemof Escherichia coli, is highly homologous to the repressor proteinsGalR, CytR, and LacI. J. Bacteriol. 171:4888-4499); malT (malA)(Bebarbouille, M., and M. Schwartz. Mutants which make more malTproduct, the activator of the maltose regulon in Escherichia coli. Mol.Gen. Genet. 178:589-595); marA (cpxB, soxQ) (Ariza, R. R., et al.Repressor mutations in the marRAB operon that activate oxidative stressgenes and multiple antibiotic resistance in Escherichia coli. J.Bacteriol. 176:143-148); marB (Berlyn, M. K. B., et al. 1996. Linkagemap of Escherichia coli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss,J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff,M. Riley, M. Schaechter, and H. E. Umbarger (eds.). Escherichia coli andSalmonella typhimurium: cellular and molecular biology, 2nd ed. AmericanSociety for Microbiology, Washington D.C.); marR (Ariza, R. R., et al.Repressor mutations in the marRAB operon that activate oxidative stressgenes and multiple antibiotic resistance in Escherichia coli. J.Bacteriol. 176:143-148); melR (Williams, J., et al. 1994. Interactionsbetween the Escherichia coli MelR transcription activator protein andoperator sequences at the melAB promoter. Biochem. J. 300:757-763); metJ(Smith, A. A., et al. 1985. Isolation and characterization of theproduct of the methionine-regulatory gene metJ of Escherichia coli K-12.Proc. Natl. Acad. Sci. 82:6104-6108; Shoeman, R., et al. 1985.Regulation of methionine synthesis in Escherichia coli: effect of metJgene product and S-adenosylmethionine on the in vitro expression of themetB, metL and metJ genes. Biochem. Biophys. Res. Commun. 133:731-739);metr (Maxon, M. E., et al. 1989. Regulation of methionine synthesis inEscherichia coli: effect of the MetR protein on the expression of themetE and metR genes. Proc. Natl. Acad. Sci. 86:85-89); mglR (R-MG)(Berlyn, M. K. B., et al. 1996. Linkage map of Escherichia coli K-12,Edition 9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin,K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, andH. E. Umbarger (eds.). Escherichia coli and Salmonella typhimurium:cellular and molecular biology, 2nd ed. American Society forMicrobiology, Washington D.C.); mhpR (Ferrandez, A., et al. 1997.Genetic characterization and expression in heterologous hosts of the3-(3-hydroxyphenyl)propionate catabolic pathway of Escherichia coliK-12. J. Bacteriol. 179:2573-2581); mhpS (Berlyn, M. K. B., et al. 1996.Linkage map of Escherichia coli K-12, Edition 9. In F. C. Neidhardt, R.Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S.Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds.).Escherichia coli and Salmonella typhimurium: cellular and molecularbiology, 2nd ed. American Society for Microbiology, Washington D.C.);micF (stc) (Aiba, H., et al. 1987. Function of micF as an antisense RNAin osmoregulatory expression of the ompF gene in Escherichia coli. J.Bacteriol. 169:3007-3012); mprA (emrR) (del Castillo, I., et al. 1990.mprA, an Escherichia coli gene that reduces growth-phase-dependentsynthesis of microcins B17 and C7 and blocks osmoinduction of proU whencloned on a high-copy-number plasmid. J. Bacteriol. 172:437-445); mtlR(Figge, R. M., et al. 1994. The mannitol repressor (MtlR) of Escherichiacoli. J. Bacteriol. 176:840-847); nagC (nagR) (Plumbridge, J. A. 1991.Repression and induction of the nag regulon of Escherichia coli K-12:the roles of nagC and nagA in maintenance of the uninduced state. Mol.Microbiol. 5:2053-3062); narL (frdR, narR) (Stewart, V. 1982.Requirement of Fnr and NarL functions for nitrate reductase expressionin Escherichia coli K-12. J. Bacteriol. 151:1320-1325; Miller, J. B., etal. 1987. Molybdenum-sensitive transcriptional regulation of the chlDlocus of Escherichia coli. J. Bacteriol. 169:1853-1860; Iuchi, S., andE. C. Lin. 1987. Molybdenum effector of fumarate reductase repressionand nitrate reductase induction in Escherichia coli. J. Bacteriol.169:3720-3725); narP (Rabin, R. S., and V. Stewart. 1993. Dual responseregulators (NarL and NarP) interact with dual sensors (NarX and NarQ) tocontrol nitrate- and nitrite-regulated gene expression in Escherichiacoli K-12. J. Bacteriol. 175:3259-3268); nhar (gene product from E.coli. (Rahav-Manor, O., et al. 1992. NhaR, a protein homologous to afamily of bacterial regulatory proteins (LysR), regulates nhaA, thesodium proton antiporter gene in Escherichia coli. J. Biol. Chem.267:10433-10438); ompR (cry, envZ, ompB) (Taylor, R. K., et al.Identification of OmpR: a positive regulatory protein controllingexpression of the major outer membrane matrix porin proteins ofEscherichia coli K-12. J. Bacteriol. 147:255-258); oxyR (mor, momR)(VanBogelen, R. A, et al. 1987. Differential induction of heat shock,SOS, and oxidation stress regulons and accumulation of nucleotides inEscherichia coli. J. Bacteriol. 169:26-32); pdhR (Haydon, D. J., et al.A mutation causing constitutive synthesis of the pyruvate dehydrogenasecomplex in Escherichia coli is located within the pdhR gene. FEBS Lett.336:43-47); phnF (Wanner, B. L., and W. W. Metcalf. 1992. Moleculargenetic studies of a 10.9-kb operon in Escherichia coli for phosphonateuptake and biodegradation. FEMS Microbiol. Lett. 79:133-139); phoB(phoRc, phoT) (Pratt, C. 1980. Kinetics and regulation of cell-freealkaline phosphatase synthesis. J. Bacteriol. 143:1265-1274); phoP(Kasahara, M., et al. 1992. Molecular analysis of the Escherichia coliphoP-phoQ operon. J. Bacteriol. 174:492-498); phoQ (Id.); phoR (R1pho,nmpB, phoR1) (Bracha, M., and E. Yagil. 1969. Genetic mapping of thephoR regulator gene of alkaline phosphatase in Escherichia coli. J. Gen.Microbiol. 59:77-81); phoU (phoT) (Nakata, A., et al. 1984. Regulationof the phosphate regulon in Escherichia coli K-12: regulation of thenegative regulatory gene phoU and identification of the gene product. J.Bacteriol. 159:979-985); poaR (Berlyn, M. K. B., et al. 1996. Linkagemap of Escherichia coli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss,J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff,M. Riley, M. Schaechter, and H. E. Umbarger (eds.). Escherichia coli andSalmonella typhimurium: cellular and molecular biology, 2nd ed. AmericanSociety for Microbiology, Washington D.C.); poxA (Chang, Y. Y., and J.E. Cronan Jr. 1982. Mapping nonselectable genes of Escherichia coli byusing transposon Tn10: location of a gene affecting pyruvate oxidase. J.Bacteriol. 151:1279-1289); proQ (Milner, J. L., and J. M. Wood. 1989.Insertion proQ220::Tn5 alters regulation of proline porter II, atransporter of proline and glycine betaine in Escherichia coli. J.Bacteriol. 171:947-951); pspA (Weiner, L., et al. 1991. Stress-inducedexpression of the Escherichia coli phage shock protein operon isdependent on sigma 54 and modulated by positive and negative feedbackmechanisms. Genes Dev. 5:1912-1923); pspB (Weiner, L., et al. 1991.Stress-induced expression of the Escherichia coli phage shock proteinoperon is dependent on sigma 54 and modulated by positive and negativefeedback mechanisms. Genes Dev. 5:1912-1923); pspC (Weiner, L., et al.1991. Stress-induced expression of the Escherichia coli phage shockprotein operon is dependent on sigma 54 and modulated by positive andnegative feedback mechanisms. Genes Dev. 5:1912-1923); pssR (Sparrow, C.P., and C. R. Raetz. 1983. A trans-acting regulatory mutation thatcauses overproduction of phosphatidylserine synthase in Escherichiacoli. J. Biol. Chem. 258:9963-9967); purR (Meng, L. M., et al. 1990.Autoregulation of PurR repressor synthesis and involvement of purR inthe regulation of purB, purC, purL, purMN and guaBA expression inEscherichia coli. Eur. J. Biochem. 187:373-379); putA (poaA) geneproduct from Salmonella enterica serotype Typhimurium (Menzel, R., andJ. Roth. 1981. Regulation of the genes for proline utilization inSalmonella typhimurium: autogenous repression by the putA gene product.J. Mol. Biol. 148:21-44); pyrI (Cunin, R., et al. 1985.Structure-function relationship in allosteric aspartatecarbamoyltransferase from Escherichia coli. I. Primary structure of apyrI gene encoding a modified regulatory subunit. J. Mol. Biol.186:707-713); rbsR (Lopilato, J. E., et al. 1984. D-ribose metabolism inEscherichia coli K-12: genetics, regulation, and transport. J.Bacteriol. 158:665-673); rcsA (Gottesman, S., et al. 1985. Regulation ofcapsular polysaccharide synthesis in Escherichia coli K-12:characterization of three regulatory genes. J. Bacteriol.162:1111-1119); rcsB (Id.); rcsC (Id.); rcsF (Grevais, F. G., and G. R.Drapeau. 1992. Identification, cloning, and characterization of rcsF, anew regulator gene for exopolysaccharide synthesis that suppresses thedivision mutation ftsZ84 in Escherichia coli K-12. J. Bacteriol.174:8016-8022); relB (Christensen, S. K., et al. 2001. RelE, a globalinhibitor of translation, is activated during nutritional stress. ProcNatl. Acad. Sci. 98:14328-14333); rfaH (sfrB) (Pradel, E., and C. A.Schnaitman. 1991. Effect of rfaH (sfrB) and temperature on expression ofrfa genes of Escherichia coli K-12. J. Bacteriol. 173:6428-6431); rhaR(Tobin, J. F., and R. F. Schleif. 1987. Positive regulation of theEscherichia coli L-rhamnose operon is mediated by the products oftandemly repeated regulatory genes. J. Mol. Biol. 196:789-799); rhaS(Id.); rnk (Berlyn, M. K. B., et al. 1996. Linkage map of Escherichiacoli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E.C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.Schaechter, and H. E. Umbarger (eds.). Escherichia coli and Salmonellatyphimurium: cellular and molecular biology, 2nd ed. American Societyfor Microbiology, Washington D.C.); rob (Skarstad, K., et al. A novelbinding protein of the origin of the Escherichia coli chromosome. J.Biol. Chem. 268:535-5370); rseA (mclA) (Missiakas, D., et al. 1997.Modulation of the Escherichia coli sigmaE (RpoE) heat-shocktranscription-factor activity by the RseA, RseB and RseC proteins. Mol.Microbiol. 24:355-371; De Las Penas, A. 1997. The sigmaE-mediatedresponse to extracytoplasmic stress in Escherichia coli is transduced byRseA and RseB, two negative regulators of sigmaE. Mol. Microbiol.24:373-385); rseB (Id.); rseC (Id.); rspA (Huisman, G. W., and T.Kolter. 1994. Sensing starvation: a homoserine lactone-dependentsignaling pathway in Escherichia coli. Science. 265:537-539); rspB(Shafqat, J., et al. An ethanol-inducible MDR ethanoldehydrogenase/acetaldehyde reductase in Escherichia coli: structural andenzymatic relationships to the eukaryotic protein forms. Eur. J.Biochem. 263:305-311); rssA (Berlyn, M. K. B., et al. 1996. Linkage mapof Escherichia coli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J.L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M.Riley, M. Schaechter, and H. E. Umbarger (eds.). Escherichia coli andSalmonella typhimurium: cellular and molecular biology, 2nd ed. AmericanSociety for Microbiology, Washington D.C.); rssB (Muffler, A., et al.1996. The response regulator RssB controls stability of the sigma(S)subunit of RNA polymerase in Escherichia coli. EMBO J. 15:1333-1339);sbaA (Berlyn, M. K. B., et al. 1996. Linkage map of Escherichia coliK-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C.C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.Schaechter, and H. E. Umbarger (eds.). Escherichia coli and Salmonellatyphimurium: cellular and molecular biology, 2nd ed. American Societyfor Microbiology, Washington D.C.); sdaC (Id.); sdiA (Sitnikov, D. M.,et al. 1996. Control of cell division in Escherichia coli: regulation oftranscription of I involves both rpoS and SdiA-mediated autoinduction.Proc. Natl. Acad. Sci. 93:336-341); serR (Theall, G., et al. 1979.Regulation of the biosynthesis of aminoacyl-tRNA synthetases and of tRNAin Escherichia coli. IV. Mutants with increased levels of leucyl- orseryl-tRNA synthetase. Mol. Gen. Genet. 169:205-211); sfsA (Takeda, K.,et al. 2001. Effects of the Escherichia coli sfsA gene on mal genesexpression and a DNA binding activity of SfsA. Biosci. Biotechnol.Biochem. 65:213-217); sfsB (nlp, sfs1) (Berlyn, M. K. B., et al. 1996.Linkage map of Escherichia coli K-12, Edition 9. In F. C. Neidhardt, R.Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S.Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds.).Escherichia coli and Salmonella typhimurium: cellular and molecularbiology, 2nd ed. American Society for Microbiology, Washington D.C.);soxR (Tsaneva, I. R., and B. Weiss. 1990. soxR, a locus governing asuperoxide response regulon in Escherichia coli K-12. J. Bacteriol.172:4197-4205); soxS (Wu, J., and B. Weiss. 1991. Two divergentlytranscribed genes, soxR and soxS, control a superoxide response regulonof Escherichia coli. J. Bacteriol. 173:2864-2871); sr1R (gutR) (Csonka,L. N., and A. J. Clark. 1979. Deletions generated by the transposon Tn10in the sr1 recA region of the Escherichia coli K-12 chromosome.Genetics. 93:321-343); tdcA (Ganduri, Y. L., et al. 1993. TdcA, atranscriptional activator of the tdcABC operon of Escherichia coli, is amember of the LysR family of proteins. Mol. Gen. Genet. 240:395-402);tdcR (Hagewood, B. T., et al. 1994. Functional analysis of the tdcABCpromoter of Escherichia coli: roles of TdcA and TdcR. J. Bacteriol.176:6241-6220); thrS (Springer, M., et al. 1985. Autogenous control ofEscherichia coli threonyl-tRNA synthetase expression in vivo. J. Mol.Biol. 185:93-104); torR (Simon, G., et al. 1994. The torR gene ofEscherichia coli encodes a response regulator protein involved in theexpression of the trimethylamine N-oxide reductase genes. J. Bacteriol.176:5601-5606); treR (Horlacher, R., and W. Boos. 1997. Characterizationof TreR, the major regulator of the Escherichia coli trehalose system.J. Biol. Chem. 272:13026-13032); trpR (Gunsalus, R. P., and C. Yanofsky.1980. Nucleotide sequence and expression of Escherichia coli trpR, thestructural gene for the trp aporepressor. Proc. Natl. Acad. Sci.77:7117-7121); tyrR (Camakaris, H., and J. Pittard. 1973. Regulation oftyrosine and phenylalanine biosynthesis in Escherichia coli K-12:properties of the tyrR gene product. J. Bacteriol. 115:1135-1144); uhpA(Kadner, R. J., and D. M. Shattuck-Eidens. 1983. Genetic control of thehexose phosphate transport system of Escherichia coli: mapping ofdeletion and insertion mutations in the uhp region. J. Bacteriol.155:1052-1061); uidR (gusR) (Novel, M., and G. Novel. 1976. Regulationof beta-glucuronidase synthesis in Escherichia coli K-12: pleiotropicconstitutive mutations affecting uxu and uidA expression. J. Bacteriol.127:418-432); uspA (Berlyn, M. K. B., et al. 1996. Linkage map ofEscherichia coli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L.Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M.Riley, M. Schaechter, and H. E. Umbarger (eds.). Escherichia coli andSalmonella typhimurium: cellular and molecular biology, 2nd ed. AmericanSociety for Microbiology, Washington D.C.); uxuR (Novel, M., and G.Novel. 1976. Regulation of beta-glucuronidase synthesis in Escherichiacoli K-12: pleiotropic constitutive mutations affecting uxu and uidAexpression. J. Bacteriol. 127:418-432); wrbA (Yang, W., et al. 1993. Astationary-phase protein of Escherichia coli that affects the mode ofassociation between the trp repressor protein and operator-bearing DNA.Proc. Natl. Acad. Sci. 90:5796-5800); xapR (pndR) (Seeger, C., et al.1995. Identification and characterization of genes (xapA, xapB, andxapR) involved in xanthosine catabolism in Escherichia coli. J.Bacteriol. 177:5506-5516); and xylR (Inouye, S., et al. 1987. Expressionof the regulatory gene xylS on the TOL plasmid is positively controlledby the xylR gene product. Proc. Natl. Acad. Sci. 84:5182-5186);

[0350] Regulatory elements, promoters and other expression elements andfactors from prokaryotes other than E. coli and B. subtilis includewithout limitation ahyRI gene product from Aeromonas hydrophila andAeromonas salmonicida (Swift, S., et al. 1997. Quorum sensing inAeromonas hydrophila and Aeromonas salmonicida: identification of theLuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserinelactone signal molecules. J. Bacteriol. 179:5271-5281); angR geneproduct from Vibrio anguillarum (Salinas, P. C., et al. 1989. Regulationof the iron uptake system in Vibrio anguillarum: evidence for acooperative effect between two transcriptional activators. Proc. Natl.Acad. Sci. 86:3529-3522); aphA gene product from Vibrio cholerae(Kovacikova, G., and K. Skorupski. 2001. Overlapping binding sites forthe virulence gene regulators AphA, AphB and cAMP-CRP at the Vibriocholerae tcpPH promoter. Mol. Microbiol. 41:393-407); aphB gene productfrom Vibrio cholerae (Kovachikova, G., and K. Skorupski. 2000.Differential activation of the tcpPH promoter by AphB determines biotypespecificity of virulence gene expression in Vibrio cholerae. J.Bacteriol. 182:3228-3238); comE gene product from Streptococcuspneumoniae (Ween, O., et al. 1999. Identification of DNA binding sitesfor ComE, a key regulator of natural competence in Streptococcuspneumoniae. Mol. Microbiol. 33:817-827); esaI gene product from Pantoeastewartii subsp. stewartii (von Bodman, S. B., et al. 1998. A negativeregulator mediates quorum-sensing control of exopolysaccharideproduction in Pantoea stewartii subsp. stewartii. Proc. Natl. Acad. Sci.95:7687-7692); esaR gene product from Pantoea stewartii subsp. stewartii(Id.); expI gene product from Erwinia chrysanthemi (Nasser, W., et al.1998. Characterization of the Erwinia chrysanthemi expI-expR locusdirecting the synthesis of two N-acyl-homoserine lactone signalmolecules. Mol. Microbiol. 29:1391-1405); expR gene product from Erwiniachrysanthemi (Id.); gacA gene product from Pseudomonas aeruginosa(Pessi, G., and D. Haas. 2001. Dual control of hydrogen cyanidebiosynthesis by the global activator GacA in Pseudomonas aeruginosaPAO1. FEMS Microbiol. Lett. 200:73-78); hapR gene product from Vibriocholerae (Jobling, M. G., and R. K. Holmes. Characterization of hapR, apositive regulator of the Vibrio cholerae HA/protease gene hap, and itsidentification as a functional homologue of the Vibrio harveyi luxRgene. Mol. Microbiol. 26:1023-1034); hlyR gene product from Vibriocholerae (von Mechow, S., et al. 1985. Mapping of a gene that regulateshemolysin production in Vibrio cholerae. J. Bacteriol. 163:799-802);hupR gene product from Vibrio vulnificus (Litwin, C. M., and J.Quackenbush. 2001. Characterization of a Vibrio vulnificus LysRhomologue, HupR, which regulates expression of the haem uptake outermembrane protein, HupA. Microb. Pathog. 31:295-307); lasR gene productfrom Pseudomonas aerugenosa (Gambella, M. J., and B. H. Igleweski. 1991.Cloning and characterization of the Pseudomonas aeruginosa lasR gene, atranscriptional activator of elastase expression. J. Bacteriol.173:3000-3009); leuO gene product from Salmonella enterica serovarTyphimurium (Fang, M., and H. Y. Wu. 1998. A promoter relay mechanismfor sequential gene activation. J. Bacteriol. 180:626-633); luxI geneproduct from Vibrio cholerae (Engebrecht, J., and M. Silverman.Nucleotide sequence of the regulatory locus controlling expression ofbacterial genes for bioluminescence. Nucleic Acids Res. 15:10455-10467);luxO gene product from Vibrio cholerae (Bassler, B. L., et al. 1994.Sequence and function of LuxO, a negative regulator of luminescence inVibrio harveyi. Mol. Microbiol. 12:403-412); luxR gene product fromVibrio cholerae (Engebrecht, J., and M. Silverman. Nucleotide sequenceof the regulatory locus controlling expression of bacterial genes forbioluminescence. Nucleic Acids Res. 15:10455-10467); phzR gene productfrom Pseudomonas aureofaciens (Pierson, L. S., et al. 1994. Phenazineantibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulatedby PhzR in response to cell density. J. Bacteriol. 176:3966-3974); rhlRgene product from Pseudomonas aeruginosa (Ochsner, U. A. et al. 1994.Isolation and characterization of a regulatory gene affectingrhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. J.Bacteriol. 176:2044-2054); rsmA gene product from Erwinia carotovorasubsp. carotovora (Cui, Y., et al. 1995. Identification of a globalrepressor gene, rsmA, of Erwinia carotovora subsp. carotovora thatcontrols extracellular enzymes, N-(3-oxohexanoyl)-L-homoserine lactone,and pathogenicity in soft-rotting Erwinia spp. J. Bacteriol.177:5108-5115); rsmB gene product from Erwinia carotovora subsp.carotovora (Cui, Y., et al. 1999. rsmC of the soft-rotting bacteriumErwinia carotovora subsp. carotovora negatively controls extracellularenzyme and harpin(Ecc) production and virulence by modulating levels ofregulatory RNA (rsmB) and RNA-binding protein (RsmA). J. Bacteriol.181:6042-6052); sirA gene product from Salmonella enterica serovarTyphimurium (Goodier, R. I., and B. M. Ahmer. 2001. SirA orthologsaffects both motility and virulence. J. Bacteriol. 183:2249-2258); tafgene product from Vibrio cholerae (Salinas, P. C., et al. 1989.Regulation of the iron uptake system in Vibrio anguillarum: evidence fora cooperative effect between two transcriptional activators. Proc. Natl.Acad. Sci. 86:3529-3522); tcpP gene product from Vibrio cholerae (Hase,C. C., and J. J. Mekalanos. 1998. TcpP protein is a positive regulatorof virulence gene expression in Vibrio cholerae. Proc. Natl. Acad. Sci.95:730-734); toxR gene product from Vibrio cholerae (Miller, V. L., andJ. J. Mekalanos. 1984. Synthesis of cholera toxin is positivelyregulated at the transcriptional level by toxR. Proc. Natl. Acad. Sci.81:3471-4375); toxS gene product from Vibrio cholerae (Miller, V. L., etal. 1989. Identification of toxS, a regulatory gene whose productenhances toxR-mediated activation of the cholera toxin promoter. J.Bacteriol. 171:1288-1293); toxT from Vibrio cholerae (Kaufman, M. R., etal. 1993. Biogenesis and regulation of the Vibrio choleraetoxin-coregulated pilus: analogies to other virulence factor secretorysystems. Gene. 126:43-49); traM gene product from Agrobacteriumtumefaciens (Faqua, C., et al. 1995. Activity of the Agrobacterium Tiplasmid conjugal transfer regulator TraR is inhibited by the product ofthe traM gene. J. Bacteriol. 177:1367-1373); traR gene product fromAgrobacterium tumefaciens (Piper, K. R., et al. 1993. Conjugation factorof Agrobacterium tumefaciens regulates Ti plasmid transfer byautoinduction. Nature. 362:448-450); vicH gene product from Vibriocholerae (Tendeng, C., et al. 2000. Isolation and characterization ofvicH, encoding a new pleiotropic regulator in Vibrio cholerae. J.Bacteriol. 182:2026-2032); vspR gene product from Vibrio cholerae(Yildiz, F. H., et al. 2001. VpsR, a Member of the Response Regulatorsof the Two-Component Regulatory Systems, Is Required for Expression ofvps Biosynthesis Genes and EPS(ETr)-Associated Phenotypes in Vibriocholerae O1 El Tor. J. Bacteriol. 183:1716-1726).

[0351] II. C. 5.b. Bacillus subtilis

[0352] Regulatory elements, promoters and other expression elements andexpression elements from B subtilis include but are not limited to abrB(Perego, M., et al. 1988. Structure of the gene for the transition stateregulator, abrB: regulator synthesis is controlled by the spo0Asporulation gene in Bacillus subtilis Mol. Microbiol. 2:698-699); acoR(Ali, N. O., et al. 2001. Regulation of the acetoin catabolic pathway iscontrolled by sigma L in Bacillus subtilis J. Bacteriol. 183:2497-2504);ahrC (Klinger, U., et al. 1995. A binding site for activation by theBacillus subtilis AhrC protein, a repressor/activator of argininemetabolism. Mol. Gen. Genet. 248:329-340); alaR (Sohenshein, A. L., J.A. Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and its closestrelatives: from genes to cells. American Society for Microbiology,Washington D.C.); alsR (Renna, M. C., et al. 1993. Regulation of theBacillus subtilis alsS, alsD, and alsR genes involved inpost-exponential-phase production of acetoin. J. Bacteriol.175:3863-3875); ansR (Sun, D., and P. Setlow. 1993. Cloning andnucleotide sequence of the Bacillus subtilis ansR gene, which encodes arepressor of the ans operon coding for L-asparaginase and L-aspartase.J. Bacteriol. 175:2501-2506); araR (Sa-Nogueira, I., and L. J. Mota.1997. Negative regulation of L-arabinose metabolism in Bacillussubtilis: characterization of the araR (araC) gene. J. Bacteriol.179:1598-1608); arfM (Marino, M., et al. 2001. Modulation of anaerobicenergy metabolism of Bacillus subtilis by arfM (ywiD). J. Bacteriol.183:6815-6821); arsR (Rosenstein, R., et al. 1992. Expression andregulation of the antimonite, arsenite, and arsenate resistance operonof Staphylococcus xylosus plasmid pSX267. J. Bacteriol. 174:3676-3683);azlB (Belitsky, B. R., et al. 1997. An lrp-like gene of Bacillussubtilis involved in branched-chain amino acid transport. J. Bacteriol.179:54485457); birA (Bower, S., et al. 1995. Cloning andcharacterization of the Bacillus subtilis birA gene encoding a repressorof the biotin operon. J. Bacteriol. 177:2572-2575); bkdR (Bebarbouille,M., et al. 1999. Role of bkdR, a transcriptional activator of thesigL-dependent isoleucine and valine degradation pathway in Bacillussubtilis J. Bacteriol. 181:2059-2066); bltR (Ahmed, M., et al. 1995. Twohighly similar multidrug transporters of Bacillus subtilis whoseexpression is differentially regulated. J. Bacteriol. 177:3904-3910);bmrR (Ahmed, M., et al. 1994. A protein that activates expression of amultidrug efflux transporter upon binding the transporter substrates. J.Biol. Chem. 269:28506-28513); ccpA (Henkin, T. M., et al. 1991.Catabolite repression of alpha-amylase gene expression in Bacillussubtilis involves a trans-acting gene product homologous to theEscherichia coli lac1 and galR repressors. Mol. Microbiol. 5:575-584);ccpB (Chauvaux, S., et al. 1998. CcpB, a novel transcription factorimplicated in catabolite repression in Bacillus subtilis. J. Bacteriol.180:491-497); ccpC (Jourlin-Castelli, C., et al. 2000. CcpC, a novelregulator of the LysR family required for glucose repression of the citBgene in Bacillus subtilis J. Mol. Biol. 295:865-878); cggR (Fillinger,S., et al. 2000. Two glyceraldehyde-3-phosphate dehydrogenases withopposite physiological roles in a nonphotosynthetic bacterium. J. Biol.Chem. 275:14031-14037); cheB (Bischoff, D. S., and G. W. Ordal. 1991.Sequence and characterization of Bacillus subtilis CheB, a homolog ofEscherichia coli CheY, and its role in a different mechanism ofchemotaxis. J. Biol. Chem. 266:12301-12305); cheY (Bischoff, D. S., etal. 1993. Purification and characterization of Bacillus subtilis CheY.Biochemistry 32:9256-9261); citR (Jin, S., and A. L. Sonenshein. 1994.Transcriptional regulation of Bacillus subtilis citrate synthase genes.J. Bacteriol. 176:4680-4690); citT (Yamamoto, H., et al. 2000. The CitSTtwo-component system regulates the expression of the Mg-citratetransporter in Bacillus subtilis Mol. Microbiol. 37:898-912); codY(Slack, F. J., et al. 1995. A gene required for nutritional repressionof the Bacillus subtilis dipeptide permease operon. Mol. Microbiol.15:689-702); comA (Nakano, M. M., and P. Zuber. 1989. Cloning andcharacterization of srfB, a regulatory gene involved in surfactinproduction and competence in Bacillus subtilis. J. Bacteriol.171:5347-5353); comK (Msadek, T., et al. 1994. MecB of Bacillussubtilis, a member of the ClpC ATPase family, is a pleiotropic regulatorcontrolling competence gene expression and growth at high temperature.Proc. Natl. Acad. Sci. 91:5788-5792); comQ (Weinrauch, Y., et al. 1991.Sequence and properties of comQ, a new competence regulatory gene ofBacillus subtilis. J. Bacteriol. 173:5685-5693); cssR (Hyyrylainen, H.L., et al. 2001. A novel two-component regulatory system in Bacillussubtilis for the survival of severe secretion stress. Mol. Microbiol.41:1159-1172); ctsR (Kruger, E., and M. Hecker. 1998. The first gene ofthe Bacillus subtilis c1pC operon, ctsR, encodes a negative regulator ofits own operon and other class III heat shock genes. J. Bacteriol.180:6681-6688); dctR (Sohenshein, A. L., J. A. Hoch, and R. Losick(eds.) 2002. Bacillus subtilis and its closest relatives: from genes tocells. American Society for Microbiology, Washington D.C.); degA(Bussey, L. B., and R. L. Switzer. 1993. The degA gene productaccelerates degradation of Bacillus subtilis phosphoribosylpyrophosphateamidotransferase in Escherichia coli J. Bacteriol. 175:6348-6353); degU(Msadek, T., et al. 1990. Signal transduction pathway controllingsynthesis of a class of degradative enzymes in Bacillus subtilis:expression of the regulatory genes and analysis of mutations in degS anddegU. J. Bacteriol. 172:824-834); deoR (Saxild, H. H., et al. 1996.Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, inductionby deoxyribonucleosides, and transcriptional regulation by thedeoR-encoded DeoR repressor protein. J. Bacteriol. 178:424-434); exuR(Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillussubtilis and its closest relatives: from genes to cells. AmericanSociety for Microbiology, Washington D.C.); frn (Cruz Ramos, H., et al.1995. Anaerobic transcription activation in Bacillus subtilis:identification of distinct FNR-dependent and -independent regulatorymechanisms. EMBO J. 14:5984-5994); fruR (Saier, M. H. Jr. 1996. CyclicAMP-independent catabolite repression in bacteria. FEMS Microbiol. Lett.138:97-103); fur (Chen, L., et al. 1993. Metalloregulation in Bacillussubtilis: isolation and characterization of two genes differentiallyrepressed by metal ions. J. Bacteriol. 175:5428-5437); gabR (Sohenshein,A. L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and itsclosest relatives: from genes to cells. American Society forMicrobiology, Washington D.C.); gerE (Holand, S. K., et al. 1987. Thepossible DNA-binding nature of the regulatory proteins, encoded byspoIID and gerE, involved in the sporulation of Bacillus subtilis J.Gen. Microbiol. 133:2381-2391); glcR (Stulke, J., et al. 2001.Characterization of glucose-repression-resistant mutants of Bacillussubtilis: identification of the glcR gene. Arch. Microbiol. 175:441449);glcT (Paulsen, I. T., et al. 1998. Characterization of glucose-specificcatabolite repression-resistant mutants of Bacillus subtilis:identification of a novel hexose:H+symporter. J. Bacteriol.180:498-504); glnR (Schreier, H. J., et al. 1989. Regulation of Bacillussubtilis glutamine synthetase gene expression by the product of the glnRgene. J. Mol. Biol. 210:51-63); glpP (Holmberg, C., and B. Rutberg.1991. Expression of the gene encoding glycerol-3-phosphate dehydrogenase(glpD) in Bacillus subtilis is controlled by antitermination. Mol.Microbiol. 5:2891-2900); gltC (Bohannon, D. E. and A. L. Sonenshein.1989. Positive regulation of glutamate biosynthesis in Bacillussubtilis. J. Bacteriol. 171:4718-4727); gltR (Belitsky, B. R., and A. L.Sonenshein. 1997. Altered transcription activation specificity of amutant form of Bacillus subtilis GltR, a LysR family member. J.Bacteriol. 179:1035-1043); gntR (Fujita, Y., and T. Fujita. 1987. Thegluconate operon gnt of Bacillus subtilis encodes its owntranscriptional negative regulator. Proc. Nadl. Acad. Sci.84:4524-4528); gutR (Ye, R., et al. 1994. Glucitol induction in Bacillussubtilis is mediated by a regulatory factor, GutR. J. Bacteriol.176:3321-3327); hpr (Perego, M., and J. A. Hoch. 1988. Sequence analysisand regulation of the hpr locus, a regulatory gene for proteaseproduction and sporulation in Bacillus subtilis. J. Bacteriol.170:2560-2567); hrcA (Schulz, A., and W. Schumann. 1996. hrcA, the firstgene of the Bacillus subtilis dnaK operon encodes a negative regulatorof class I heat shock genes. J. Bacteriol. 178:1088-1093); hutP (Oda,M., et al. 1992. Analysis of the transcriptional activity of the hutpromoter in Bacillus subtilis and identification of a cis-actingregulatory region associated with catabolite repression downstream fromthe site of transcription. Mol. Microbiol. 6:2573-2582); hxlR(Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillussubtilis and its closest relatives: from genes to cells. AmericanSociety for Microbiology, Washington D.C.); iolR (Yoshida, K. I., et al.1999. Interaction of a repressor and its binding sites for regulation ofthe Bacillus subtilis iol divergon. J. Mol. Biol. 285:917-929); kdgR(Pujic, P., et al. 1998. The kdgRKAT operon of Bacillus subtilis:detection of the transcript and regulation by the kdgR and ccpA genes.Microbiology. 144:3111-3118); kipR (Sohenshein, A. L., J. A. Hoch, andR. Losick (eds.) 2002. Bacillus subtilis and its closest relatives: fromgenes to cells. American Society for Microbiology, Washington D.C.);lacR (Errington, J., and C. H. Vogt. 1990. Isolation andcharacterization of mutations in the gene encoding an endogenousBacillus subtilis beta-galactosidase and its regulator. J. Bacteriol.172:488-490); levR (Bebarbouille, M., et al. 1991. The transcriptionalregulator LevR of Bacillus subtilis has domains homologous to both sigma54- and phosphotransferase system-dependent regulators. Proc. natl.Acad. Sci. 88:2212-2216); lexA (Lovett, C. M. Jr., and J. W. Roberts.1985. Purification of a RecA protein analogue from Bacillus subtilis. J.Biol. Chem. 260:3305-3313); licR (Tobisch, S., et a. 1997.Identification and characterization of a new beta-glucoside utilizationsystem in Bacillus subtilis. J. Bacteriol. 179:496-506); licT (Le Coq,D., et al. 1995. New beta-glucoside (bgl) genes in Bacillus subtilis:the bglP gene product has both transport and regulatory functionssimilar to those of BglF, its Escherichia coli homolog. J. Bacteriol.177:1527-1535); lmrA (Kumano, M., et al. 1997. A 32 kb nucleotidesequence from the region of the lincomycin-resistance gene (22degrees-25 degrees) of the Bacillus subtilis chromosome andidentification of the site of the lin-2 mutation. Microbiology.143:2775-2782); lrpA gene product from Pyrococcus furiosus (Brinkman, A.B., et al. 2000. An Lrp-like transcriptional regulator from the archaeonPyrococcus furiosus is negatively autoregulated. J. Biol. Chem.275:38160-38169); lrpB (Sohenshein, A. L., J. A. Hoch, and R. Losick(eds.) 2002. Bacillus subtilis and its closest relatives: from genes tocells. American Society for Microbiology, Washington D.C.); lrpC(Beloin, C., et al. 1997. Characterization of an lrp-like (lrpC) genefrom Bacillus subtilis. Mol. Gen. Genet. 256:63-71); lytR (Huang, X.,and J. D. Helmann. 1998. Identification of target promoters for theBacillus subtilis sigma X factor using a consensus-directed search. J.Mol. Biol. 279:165-173); lytT (Sohenshein, A. L., J. A. Hoch, and R.Losick (eds.) 2002. Bacillus subtilis and its closest relatives: fromgenes to cells. American Society for Microbiology, Washington D.C.);manR gene product from Listeria monocytogenes (Dalet, K., et al. 2001. Asigma(54)-dependent PTS permease of the mannose family is responsiblefor sensitivity of Listeria monocytogenes to mesentericin Y105.Microbiology. 147:3263-3269); mntR (Que, Q., and J. D. Helmann. 2000.Manganese homeostasis in Bacillus subtilis is regulated by MntR, abifunctional regulator related to the diphtheria toxin repressor familyof proteins. Mol. Microbiol. 35:1454-1468); msmR gene product fromStreptococcus mutans (Russell, R. R., et al. 1992. A bindingprotein-dependent transport system in Streptococcus mutans responsiblefor multiple sugar metabolism. J. Biol. Chem. 267:4631-4637); mta(Baranova, N. N., et al. 1999. Mta, a global MerR-type regulator of theBacillus subtilis multidrug-efflux transporters. Mol. Microbiol.31:1549-1559); mtlR (Henstra, S. A., et al. 1999. The Bacillusstearothermophilus mannitol regulator, MtlR, of the phosphotransferasesystem. A DNA-binding protein, regulated by HPr and iicbmtl-dependentphosphorylation. J. Biol. Chem. 274:4754-4763); mtrB (Gollnick, P., etal. 1990. The mtr locus is a two-gene operon required for transcriptionattenuation in the trp operon of Bacillus subtilis. Proc. Natl. Acad.Sci. 87:8726-8730); nhaX (Sohenshein, A. L., J. A. Hoch, and R. Losick(eds.) 2002. Bacillus subtilis and its closest relatives: from genes tocells. American Society for Microbiology, Washington D.C.); toxR geneproduct from Vibrio cholerae (Miller, V. L., and J. J. Mekalanos. 1984.Synthesis of cholera toxin is positively regulated at thetranscriptional level by toxR. Proc. Natl. Acad. Sci. 81:3471-3475);padR gene product from Pediococcus pentosaceus (Barthelmebs, L., et al.2000. Inducible metabolism of phenolic acids in Pediococcus pentosaceusis encoded by an autoregulated operon which involves a new class ofnegative transcriptional regulator. J. Bacteriol. 182:6724-6731); paiA(Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillussubtilis and its closest relatives: from genes to cells. AmericanSociety for Microbiology, Washington D.C.); paiB (Id.); perA (Id.); phoP(Birkey, S. M., et al. 1994. A pho regulon promoter induced undersporulation conditions. Gene. 147:95-100); pksA (Sohenshein, A. L., J.A. Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and its closestrelatives: from genes to cells. American Society for Microbiology,Washington D.C.); pucR (Schultz, A. C., et al. 2001. Functional analysisof 14 genes that constitute the purine catabolic pathway in Bacillussubtilis and evidence for a novel regulon controlled by the PucRtranscription activator. J. Bacteriol. 183:3293-3302); purR (Weng, M.,et al. 1995. Identification of the Bacillus subtilis pur operonrepressor. Proc. Natl. Acad. Sci. 92:7455-7459); pyrR (Martinussen, J.,et al. 1995. Two genes encoding uracil phosphoribosyltransferase arepresent in Bacillus subtilis. J. Bacteriol. 177:271-274); rbsR(Rodionov, D. A., et al. 2001. Transcriptional regulation of pentoseutilisation systems in the Bacillus/Clostridium group of bacteria. FEMSMicrobiol. Lett. 205:305-314); resD (Suin, G., et al. 1996. Regulatorsof aerobic and anaerobic respiration in Bacillus subtilis. J. Bacteriol.178:1374-1385); rocR (Gardan, R., et al. 1997. Role of thetranscriptional activator RocR in the arginine-degradation pathway ofBacillus subtilis. Mol. Microbiol. 24:825-837); rsiX (Tortosa, P., etal. 2000. Characterization of ylbF, a new gene involved in competencedevelopment and sporulation in Bacillus subtilis. Mol. Microbiol.35:1110-1119); sacT(Debarbouille, M., et al. 1990. The sacT generegulating the sacPA operon in Bacillus subtilis shares strong homologywith transcriptional antiterminators. J. Bacteriol. 172:3966-3973); sacV(Wong, S. L., et al. 1988. Cloning and nucleotide sequence of senN, anovel ‘Bacillus natto’ (B. subtilis) gene that regulates expression ofextracellular protein genes. J. Gen. Microbiol. 134:3269-3276); sacY(Steinmetz, M., et al 1989. Induction of saccharolytic enzymes bysucrose in Bacillus subtilis: evidence for two partially interchangeableregulatory pathways. J. Bacteriol. 171:1519-1523); senS (Wang, L. F.,and R. H. Dori. 1990. Complex character of senS, a novel gene regulatingexpression of extracellular-protein genes of Bacillus subtilis. J.Bacteriol. 172:1939-1947); sinR (Bai, U., et al. 1993. SinI modulatesthe activity of SinR, a developmental switch protein of Bacillussubtilis, by protein-protein interaction. Genes Dev. 7:139-148); slr(Asayama, M., et al. 1998. Translational attenuation of the Bacillussubtilis spo0B cistron by an RNA structure encompassing the initiationregion. Nucleic Acids Res. 26:824-830); splA (Fajardo-Cavazos, P., andW. L. Nicholson. 2000. The TRAP-like SplA protein is a trans-actingnegative regulator of spore photoproduct lyase synthesis during Bacillussubtilis sporulation. J. Bacteriol. 182:555-560); spo0A (Smith, I., etal. 1991. The role of negative control in sporulation. Res. Microbiol.142:831-839); spo0F (Lewandoski, M., et al. 1986. Transcriptionalregulation of the spo0F gene of Bacillus subtilis. J. Bacteriol.168:870-877); spoIIID (Kunkel, B., et al. 1989. Temporal and spatialcontrol of the mother-cell regulatory gene spoIIID of Bacillus subtilis.Genes. Dev. 3:1735-1744); spoVT (Bagyan, I, et al. 1996. Acompartmentalized regulator of developmental gene expression in Bacillussubtilis. J. Bacteriol. 178:4500-4507); tenA (Pang, A. S., et al. 1991.Cloning and characterization of a pair of novel genes that regulateproduction of extracellular enzymes in Bacillus subtilis. J. Bacteriol.173:46-54); tenl (Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.)2002. Bacillus subtilis and its closest relatives: from genes to cells.American Society for Microbiology, Washington D.C.); tnrA (Wray, L. V.,Jr., et al. 1996. TnrA, a transcription factor required for globalnitrogen regulation in Bacillus subtilis. Proc. Natl. Acad. Sci.93:8841-8845); treR (Schock, F., and M. K. Dahl. 1996. Expression of thetre operon of Bacillus subtilis 168 is regulated by the repressor TreR.J. Bacteriol. 178:4576-4581); xre (McDonnell, G. E., et al. 1994.Genetic control of bacterial suicide: regulation of the induction ofPBSX in Bacillus subtilis. J. Bacteriol. 176:5820-5830); xylR geneproduct from Bacillus megaterium (Rygus, T., et al. 1991. Molecularcloning, structure, promoters and regulatory elements for transcriptionof the Bacillus megaterium encoded regulon for xylose utilization. Arch.Microbiol. 155:535:542); yacF (Sohenshein, A. L., J. A. Hoch, and R.Losick (eds.) 2002. Bacillus subtils and its closest relatives: fromgenes to cells. American Society for Microbiology, Washington D.C.); andzur (Gaballa, A., and J. D. Helmann. 1998. Identification of azinc-specific metalloregulatory protein, Zur, controlling zinc transportoperons in Bacillus subtilis. J. Bacteriol. 180:5815-5821).

[0353] II.C.5.c. Other Eubacteria

[0354] Regulatory elements, promoters and other expression elements andfactors from prokaryotes other than E. coli and B. subtilis includewithout limitation ahyRI gene product from Aeromonas hydrophila andAeromonas salmonicida (Swift, S., et al. 1997. Quorum sensing inAeromonas hydrophila and Aeromonas salmonicida: identification of theLuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserinelactone signal molecules. J. Bacteriol. 179:5271-5281); angR geneproduct from Vibrio anguillarum (Salinas, P. C., et al. 1989. Regulationof the iron uptake system in Vibrio anguillarum: evidence for acooperative effect between two transcriptional activators. Proc. Natl.Acad. Sci. 86:3529-3522); aphA gene product from Vibrio cholerae(Kovacikova, G., and K. Skorupski. 2001. Overlapping binding sites forthe virulence gene regulators AphA, AphB and cAMP-CRP at the Vibriocholerae tcpPH promoter. Mol. Microbiol. 41:393-407); aphB gene productfrom Vibrio cholerae (Kovachikova, G., and K. Skorupski. 2000.Differential activation of the tcpPH promoter by AphB determines biotypespecificity of virulence gene expression in Vibrio cholerae. J.Bacteriol. 182:3228-3238); comE gene product from Streptococcuspneumoniae (Ween, O., et al. 1999. Identification of DNA binding sitesfor ComE, a key regulator of natural competence in Streptococcuspneumoniae. Mol. Microbiol. 33:817-827); esaI gene product from Pantoeastewartii subsp. stewartii (von Bodman, S. B., et al. 1998. A negativeregulator mediates quorum-sensing control of exopolysaccharideproduction in Pantoea stewartii subsp. stewartii. Proc. Natl. Acad. Sci.95:7687-7692); esaR gene product from Pantoea stewartii subsp. stewartii(Id.); expI gene product from Erwinia chrysanthemi (Nasser, W., et al.1998. Characterization of the Erwinia chrysanthemi expI-expR locusdirecting the synthesis of two N-acyl-homoserine lactone signalmolecules. Mol. Microbiol. 29:1391-1405); expR gene product from Erwiniachrysanthemi (Id.); gacA gene product from Pseudomonas aeruginosa(Pessi, G., and D. Haas. 2001. Dual control of hydrogen cyanidebiosynthesis by the global activator GacA in Pseudomonas aeruginosaPAO1. FEMS Microbiol. Lett. 200:73-78); hapR gene product from Vibriocholerae (Jobling, M. G., and R. K. Holmes. Characterization of hapR, apositive regulator of the Vibrio cholerae HA/protease gene hap, and itsidentification as a functional homologue of the Vibrio harveyi luxRgene. Mol. Microbiol. 26:1023-1034); hlyR gene product from Vibriocholerae (von Mechow, S., et al. 1985. Mapping of a gene that regulateshemolysin production in Vibrio cholerae. J. Bacteriol. 163:799-802);hupR gene product from Vibrio vulnificus (Litwin, C. M., and J.Quackenbush. 2001. Characterization of a Vibrio vulnificus LysRhomologue, HupR, which regulates expression of the haem uptake outermembrane protein, HupA. Microb. Pathog. 31:295-307); lasR gene productfrom Pseudomonas aerugenosa (Gambella, M. J., and B. H. Igleweski. 1991.Cloning and characterization of the Pseudomonas aeruginosa lasR gene, atranscriptional activator of elastase expression. J. Bacteriol.173:3000-3009); leuO gene product from Salmonella enterica serovarTyphimurium (Fang, M., and H. Y. Wu. 1998. A promoter relay mechanismfor sequential gene activation. J. Bacteriol. 180:626-633); luxI geneproduct from Vibrio cholerae (Engebrecht, J., and M. Silverman.Nucleotide sequence of the regulatory locus controlling expression ofbacterial genes for bioluminescence. Nucleic Acids Res. 15:10455-10467);luxO gene product from Vibrio cholerae (Bassler, B. L., et al. 1994.Sequence and function of LuxO, a negative regulator of luminescence inVibrio harveyi. Mol. Microbiol. 12:403-412); luxR gene product fromVibrio cholerae (Engebrecht, J., and M. Silverman. Nucleotide sequenceof the regulatory locus controlling expression of bacterial genes forbioluminescence. Nucleic Acids Res. 15:10455-10467); phzR gene productfrom Pseudomonas aureofaciens (Pierson, L. S., et al. 1994. Phenazineantibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulatedby PhzR in response to cell density. J. Bacteriol. 176:3966-3974); rhlRgene product from Pseudomonas aeruginosa (Ochsner, U. A. et al. 1994.Isolation and characterization of a regulatory gene affectingrhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. J.Bacteriol. 176:2044-2054); rsmA gene product from Erwinia carotovorasubsp. carotovora (Cui, Y., et al. 1995. Identification of a globalrepressor gene, rsmA, of Erwinia carotovora subsp. carotovora thatcontrols extracellular enzymes, N-(3-oxohexanoyl)-L-homoserine lactone,and pathogenicity in soft-rotting Erwinia spp. J. Bacteriol.177:5108-5115); rsmB gene product from Erwinia carotovora subsp.carotovora (Cui, Y., et al. 1999. rsmC of the soft-rotting bacteriumErwinia carotovora subsp. carotovora negatively controls extracellularenzyme and harpin(Ecc) production and virulence by modulating levels ofregulatory RNA (rsmB) and RNA-binding protein (RsmA). J. Bacteriol.181:6042-6052); sirA gene product from Salmonella enterica serovarTyphimurium (Goodier, R. I., and B. M. Ahmer. 2001. SirA orthologsaffects both motility and virulence. J. Bacteriol. 183:2249-2258); tafgene product from Vibrio cholerae (Salinas, P. C., et al. 1989.Regulation of the iron uptake system in Vibrio anguillarum: evidence fora cooperative effect between two transcriptional activators. Proc. Natl.Acad. Sci. 86:3529-3522); tcpP gene product from Vibrio cholerae (Hase,C. C., and J. J. Mekalanos. 1998. TcpP protein is a positive regulatorof virulence gene expression in Vibrio cholerae. Proc. Natl. Acad. Sci.95:730-734); toxR gene product from Vibrio cholerae (Miller, V. L., andJ. J. Mekalanos. 1984. Synthesis of cholera toxin is positivelyregulated at the transcriptional level by toxR. Proc. Natl. Acad. Sci.81:3471-4375); toxS gene product from Vibrio cholerae (Miller, V. L., etal. 1989. Identification of toxS, a regulatory gene whose productenhances toxR-mediated activation of the cholera toxin promoter. J.Bacteriol. 171:1288-1293); toxT from Vibrio cholerae (Kaufman, M. R., etal. 1993. Biogenesis and regulation of the Vibrio choleraetoxin-coregulated pilus: analogies to other virulence factor secretorysystems. Gene. 126:43-49); traM gene product from Agrobacteriumtumefaciens (Faqua, C., et al. 1995. Activity of the Agrobacterium Tiplasmid conjugal transfer regulator TraR is inhibited by the product ofthe traM gene. J. Bacteriol. 177:1367-1373); traR gene product fromAgrobacterium tumefaciens (Piper, K. R., et al. 1993. Conjugation factorof Agrobacterium tumefaciens regulates Ti plasmid transfer byautoinduction. Nature. 362:448-450); vicH gene product from Vibriocholerae (Tendeng, C., et al. 2000. Isolation and characterization ofvicH, encoding a new pleiotropic regulator in Vibrio cholerae. J.Bacteriol. 182:2026-2032); vspR gene product from Vibrio cholerae(Yildiz, F. H., et al. 2001. VpsR, a Member of the Response Regulatorsof the Two-Component Regulatory Systems, Is Required for Expression ofvps Biosynthesis Genes and EPS(ETr)-Associated Phenotypes in Vibriocholerae O1 El Tor. J. Bacteriol. 183:1716-1726); gadR gene product fromLactococcus lactis (Sanders, J. W., et al. 1997. A chloride-induciblegene expression cassette and its use in induced lysis of Lactococcuslactis. Appl. Environ. Microbiol. 63:4877-4882); hrpB gene product fromPseudomonas solanacearum (Van Gijsegem, F., et al. 1995. The hrp genelocus of Pseudomonas solanacearum, which controls the production of atype III secretion system, encodes eight proteins related to componentsof the bacterial flagellar biogenesis complex. Mol. Microbiol.15:1095-1114); carotovora subsp. carotovora (Cui, Y., et al. 1995.Identification of a global repressor gene, rsmA, of Erwinia carotovorasubsp. carotovora that controls extracellular enzymes,N-(3-oxohexanoyl)-L-homoserine lactone, and pathogenicity insoft-rotting Erwinia spp. J. Bacteriol. 177:5108-5115); rsmB geneproduct from Erwinia carotovora subsp. carotovora (Cui, Y., et al. 1999.rsmC of the soft-rotting bacterium Erwinia carotovora subsp. carotovoranegatively controls extracellular enzyme and harpin(Ecc) production andvirulence by modulating levels of regulatory RNA (rsmB) and RNA-bindingprotein (RsmA). J. Bacteriol. 181:6042-6052); sirA gene product fromSalmonella enterica serovar Typhimurium (Goodier, R. I., and B. M.Ahmer. 2001. SirA orthologs affects both motility and virulence. J.Bacteriol. 183:2249-2258); taf gene product from Vibrio cholerae(Salinas, P. C., et al. 1989. Regulation of the iron uptake system inVibrio anguillarum: evidence for a cooperative effect between twotranscriptional activators. Proc. Natl. Acad. Sci. 86:3529-3522); tcpPgene product from Vibrio cholerae (Hase, C. C., and J. J. Mekalanos.1998. TcpP protein is a positive regulator of virulence gene expressionin Vibrio cholerae. Proc. Natl. Acad. Sci. 95:730-734); toxR geneproduct from Vibrio cholerae (Miller, V. L., and J. J. Mekalanos. 1984.Synthesis of cholera toxin is positively regulated at thetranscriptional level by toxR. Proc. Natl. Acad. Sci. 81:3471-4375);toxS gene product from Vibrio cholerae (Miller, V. L., et al. 1989.Identification of toxS, a regulatory gene whose product enhancestoxR-mediated activation of the cholera toxin promoter. J. Bacteriol.171:1288-1293); toxT from Vibrio cholerae (Kaufman, M. R., et al. 1993.Biogenesis and regulation of the Vibrio cholerae toxin-coregulatedpilus: analogies to other virulence factor secretory systems. Gene.126:43-49); traM gene product from Agrobacterium tumefaciens (Faqua, C.,et al. 1995. Activity of the Agrobacterium Ti plasmid conjugal transferregulator TraR is inhibited by the product of the traM gene. J.Bacteriol. 177:1367-1373); traR gene product from Agrobacteriumtumefaciens (Piper, K. R., et al. 1993. Conjugation factor ofAgrobacterium tumefaciens regulates Ti plasmid transfer byautoinduction. Nature. 362:448-450); vicH gene product from Vibriocholerae (Tendeng, C., et al. 2000. Isolation and characterization ofvicH, encoding a new pleiotropic regulator in Vibrio cholerae. J.Bacteriol. 182:2026-2032); vspR gene product from Vibrio cholerae(Yildiz, F. H., et al. 2001. VpsR, a Member of the Response Regulatorsof the Two-Component Regulatory Systems, Is Required for Expression ofvps Biosynthesis Genes and EPS(ETr)-Associated Phenotypes in Vibriocholerae O1 El Tor. J. Bacteriol. 183:1716-1726); lrpA gene product fromPyrococcus furiosus (Brinkman, A. B., et al. 2000. An Lrp-liketranscriptional regulator from the archaeon Pyrococcus furiosus isnegatively autoregulated. J. Biol. Chem. 275:38160-38169); manR geneproduct from Listeria monocytogenes (Dalet, K., et al. 2001. Asigma(54)-dependent PTS permease of the mannose family is responsiblefor sensitivity of Listeria monocytogenes to mesentericin Y105.Microbiology. 147:3263-3269); msmR gene product from Streptococcusmutans (Russell, R. R., et al. 1992. A binding protein-dependenttransport system in Streptococcus mutans responsible for multiple sugarmetabolism. toxR gene product from Vibrio cholerae (Miller, V. L., andJ. J. Mekalanos. 1984. Synthesis of cholera toxin is positivelyregulated at the transcriptional level by toxR. Proc. Natl. Acad. Sci.81:3471-3475); padR gene product from Pediococcus pentosaceus(Barthelmebs, L., et al. 2000. Inducible metabolism of phenolic acids inPediococcus pentosaceus is encoded by an autoregulated operon whichinvolves a new class of negative transcriptional regulator. J.Bacteriol. 182:6724-6731); purR (Weng, M., et al. 1995); and xylR geneproduct from Bacillus megaterium (Rygus, T., et al. 1991. Molecularcloning, structure, promoters and regulatory elements for transcriptionof the Bacillus megaterium encoded regulon for xylose utilization. Arch.Microbiol. 155:535:542).

[0355] II. C.5.d. Bacteriophage and Transposable Elements

[0356] Regulatory elements, promoters and other expression elements frombacteriophage and transposable elements include without limitation cIgene product from bacteriophage lambda mation and/or segregatedminicells (Reichardt, L. F. 1975. Control of bacteriophage lambdarepressor synthesis: regulation of the maintenance pathway of the croand cI products. J. Mol. Biol. 93:289-309); (Love, C. A., et al. 1996.Stable high-copy-number bacteriophage lambda promoter vectors foroverproduction of proteins in Escherichia coli. Gene. 176:49-53); the c2gene product from bacteriophage P22 (Gough, M., and S. Tokuno. 1975.Further structural and functional analogies between the repressorregions of phages P22 and lambda. Mol. Gen. Genet. 138:71-79); the crogene from bacteriophage lambda (Reichardt, L. F. 1975. Control ofbacteriophage lambda repressor synthesis: regulation of the maintenancepathway of the cro and cI products. J. Mol. Biol. 93:289-309); the antgene from bacteriophage P22 (Youderian, P. et al. 1982. Sequencedeterminants of promotor activity. Cell. 30:843-853); the mnt gene frombacteriophage P22 (Gough, M. 1970. Requirement for a functional intproduct in temperature inductions of prophage P22 ts mnt. J. Virol.6:320-325; Prell, H. H. 1978. Ant-mediated transactivation of earlygenes in Salmonella prophage P22 by superinfecting virulent P22 mutants.Mol. Gen. Genet. 164:331-334); the tetR gene product from the TetRfamily of bacterial regulators or homologues of this gene or geneproduct found in Tn10 and other members of the bacteriophage, animalvirus, Eubacteria, Eucarya or Archaea may be employed to increase theefficiency of gene expression and protein production in parent cellsprior to minicell formation and/or segregated minicells (Moyed, H. S.,and K. P. Bertrand. 1983. Mutations in multicopy Tn10 tet plasmids thatconfer resistance to inhibitory effects of inducers of tet geneexpression. J. Bacteriol. 155:557-564); the mnt gene product frombacteriophage SP6 mation and/or segregated minicells (Mead, D. A., etal. 1985. Single stranded DNA SP6 promoter plasmids for engineeringmutant RNAs and proteins: synthesis of a ‘stretched’ preproparathyroidhormone. Nucleic Acids Res. 13:1103-1118); and the mnt gene product frombacteriophage T7 mation and/or segregated minicells (Steen, R., et al.1986. T7 RNA polymerase directed expression of the Escherichia coli rrnBoperon. EMBO J. 5:1099-1103).

[0357] II.C.5.e. Use of Site-Specific Recombination in ExpressionSystems

[0358] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inminicells. By way of non-limiting example, these techniques may includemodification of endogenous and/or exogenous regulatory elementsresponsible for activation and/or repression of proteins to be expressedfrom chromosomal and/or plasmid expression vectors. By way ofnon-limiting example, this system may be applied to any of the aboveregulatory elements/systems. Specifically, each of the above mentionedregulatory systems may be constructed such that the promotor regions areoriented in a direction away from the gene to be expressed, or each ofthe above mentioned gene(s) to be expressed may be constructed such thatthe gene(s) to be expressed is oriented in a direction away from theregulatory region promotor. Constructed in this system is a methodologydependent upon site-specific genetic recombination for inversion andinduction of the gene of interest (Backman, K., et al. 1984. Use ofsynchronous site-specific recombination in vivo to regulate geneexpression. Bio/Technology 2:1045-1049; Balakrishnan, R., et al. 1994. Agene cassette for adapting Escherichia coli strains as hosts foratt-Int-mediated rearrangement and pL expression vectors. Gene138:101-104; Hasan, N., and W. Szybalaki. 1987. Control of cloned geneexpression by promoter inversion in vivo: construction of improvedvectors with a multiple cloning site and the Ptac promotor. Gene56:145-151; Wulfing, C., and A. Pluckthun. 1993. A versatile and highlyrepressible Escherichia coli expression system based on invertiblepromoters: expression of a gene encoding a toxic gene product. Gene136:199-203). These invertible promoters and/or gene regions will allowtight regulation of potentially toxic protein products. By way ofnon-limiting example, these systems may be derived from bacteriophagelambda, bacteriophage Mu, and/or bacteriophage P22. In any of thesepotential systems, regulation of the recombinase may be regulated by anyof the regulatory systems discussed in section II.C.5 and elsewhereherein.

[0359] II.C.5.e. Use of Copy Number Control Switches

[0360] A method that can be used to increase the efficiency of geneexpression and protein production in minicells involves the modificationof endogenous and/or introduction of exogenous genetic expressionsystems such that the number of copies of a gene encoding a protein tobe expressed can be modulated. Copy number control systems compriseelements designed to modulate copy number in a controlled fashion.

[0361] In an exemplary mode, copy number is controlled to decrease theeffects of “leaky” (uninduced) expression of toxic gene products. Thisallows one to maintain the integrity of a potentially toxic gene productduring processes such as cloning, culture maintenance, and periods ofgrowth prior to minicell-induction. That is, decreasing the copy numberof a gene is expected to decrease the opportunity for mutationsaffecting protein expression and/or function to arise. Immediately priorto, during and/or after minicell formation, the copy number may beincreased to optimize the gene dosage in minicells as desired.

[0362] The replication of eubacterial plasmids is regulated by a numberof factors, some of which are contained within the plasmid, others ofwhich are located on the chromosome. For reviews, see del Solar, G., etal. 2000. Plasmid copy number control: an ever-growing story. MolMicrobiol. 37:492-500; del Solar, G., et al. 1998. Replication andcontrol of circular bacterial plasmids. Microbiol Mol Biol Rev.62:434-64; and Filutowicz, M., et al. 1987. DNA and protein interactionsin the regulation of plasmid replication. J Cell Sci Suppl. 7:15-31.

[0363] By way of non-limiting example, the pcnB gene product, thewildtype form of which promotes increased ColE1 plasmid copy number(Soderbom, F., et al. 1997. Regulation of plasmid R1 replication: PcnBand RNase E expedite the decay of the antisense RNA, CopA. Mol.Microbiol. 26:493-504), is modulated; and/or mutant forms of the pcnBgene are introduced into a cell. In an exemplary cell type that may beused in the methods of the invention, the wildtype pcnB chromosomal geneis replaced with a mutant pcnB80 allele (Lopilato, J., et al. 1986.Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB,reduce plasmid copy number of pBR322 and its derivatives. Mol. Gen.Genet. 205:285-290). In such cells the copy number of a ColE1-derivedplasmid is decreased. The cell may further comprise an expressionelement comprising an inducible promoter operably linked to an ORFencoding the wild-type pcnB. Because the wild-type pcnB gene is dominantto the mutant pcnB80 gene, and because the wild-type pcnB gene productpromotes increased ColE1 plasmid copy number, induction of a wild-typepcnB in the pcnB80 background will increase the plasmid copy number ofColE1-derived plasmids. Such copy number control systems may beexpressed from the chromosome and/or plasmid to maintain either low orhigh plasmid copy number in the absence of induction. Other non-limitingexamples of gene and/or gene products that may be employed in copynumber control systems for ColE1-based replicons include genes orhomologs of genes encoding RNA I, RNA II, rop, RNAse H, enzymes involvedin the process of polyadenylation, RNAse E, DNA polymerase I, and DNApolymerase III.

[0364] In the case of IncFII-derived replicons, non-limiting examples ofgene and/or gene products that may be employed in copy number controlsystems to control plasmid copy include genes or homologs of the copA,copB, repA, and repB genes. Copy number control systems may additionallyor alternatively include manipulation of repC, trfA, dnaA, dnaB, dnaC,seqA, genes protein Pi, genes encoding HU protein subunits (hupA, hupB)and genes encoding IHF subunits.

[0365] Other elements may also be included to optimize these plasmidcopy number control systems. Such additional elements may include theaddition or deletion of iteron nucleic acid sequences (Chattoraj, D. K.2000. Control of plasmid DNA replication by iterons: no longerparadoxical. Mol. Microbiol. 37:467-476), and modification of chaperoneproteins involved in plasmid replication (Konieczny, I., et al. 1997.The replication initiation protein of the broad-host-range plasmid RK2is activated by the ClpX chaperone. Proc Natl Acad Sci USA94:14378-14382).

[0366] II.C.6. Transportation of Inducible and Inhibitory Compounds

[0367] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inminicells. By way of non-limiting example, these techniques may includeutilization and/or modification of factors and systems that modulate thetransport of compounds, including but not limited to inducers and/orinhibitors of expression elements that control expression of a gene in aparent cell prior to minicell formation and/or in segregated minicells.Such manipulations may result in increased or decreased production,and/or changes in the intramolecular and intermolecular functions, of aprotein in a minicell or its parent cell. The techniques may be employedto increase the efficiency of gene expression and protein production inparent cells prior to minicell formation and/or in segregated minicells.

[0368]

[0369] II.C.6.a. Escherichia coli Genes

[0370] By way of non-limiting example, manipulation of the abpS gene orgene product from E. coli, or homologs of this gene or gene productfound in other members of the Prokaryotes, Eukaryotes, Archaebacteriaand/or organelles (e.g., mitochondria, chloroplasts, plastids and thelike) may be employed to increase the efficiency of gene expression andprotein production in parent cells prior to minicell formation and/or insegregated minicells (Celis, R. T. 1982. Mapping of two loci affectingthe synthesis and structure of a periplasmic protein involved inarginine and ornithine transport in Escherichia coli K-12. J. Bacteriol.151(3): 1314-9).

[0371] In addition to abpS, other exemplary E. coli genes encodingfactors involved in the transport of inducers, inhibitors and othercompounds include, but are not limited to, araE (Khlebnikov, A., et al.2001. Homogeneous expression of the P(BAD) promoter in Escherichia coliby constitutive expression of the low-affinity high-capacity AraEtransporter. Microbiology. 147(Pt 12):3241-7); araG (Kehres, D. G., andHogg, R. W. 1992. Escherichia coli K12 arabinose-binding protein mutantswith altered transport properties. Protein Sci. 1(12): 1652-60); araH(Id.); argP (Celis, R. T. 1999. Repression and activation of argininetransport genes in Escherichia coli K 12 by the ArgP protein. J. MolBiol. 17;294(5):1087-95); aroT(aroR, trpR) (Edwards, R. M., and Yudkin,M. D. 1982. Location of the gene for the low-affinitytryptophan-specific permease of Escherichia coli. Biochem. J.204(2):617-9); artI (Wissenbach, U., et al. 1995. A third periplasmictransport system for L-arginine in Escherichia coli: molecularcharacterization of the artPIQMJ genes, arginine binding and transport.Mol. Microbiol. 17(4):675-86); artJ (Id.); artM (Id.); artP (Id.); artQ(Id.); bioP (bir, birB) (Campbell, A., et al. Biotin regulatory (bir)mutations of Escherichia coli. 1980. J. Bacteriol. 142(3):1025-8); brnQ(hrbA) (Yamato, I., and Anraku, Y. 1980. Genetic and biochemical studiesof transport systems for branched-chain amino acids in Escherichia coliK-12: isolation and properties of mutants defective inleucine-repressible transport activities. J. Bacteriol. 144(1):36-44);brnR (Id.); brnS (Id.); brnT (Id.); btuC (Friedrich, M. J., et al. 1986.Nucleotide sequence of the btuCED genes involved in vitamin B12transport in Escherichia coli and homology with components ofperiplasmic-binding-protein-dependent transport systems. J. Bacteriol.167(3):928-34); btuD (Id.) (Friedrich, M. J., et al. 1986. Nucleotidesequence of the btuCED genes involved in vitamin B12 transport inEscherichia coli and homology with components ofperiplasmic-binding-protein-dependent transport systems. J. Bacteriol.167(3):928-34); caiT (Eichler, K. 1994. Molecular characterization ofthe cai operon necessary for carnitine metabolism in Escherichia coli.Mol. Microbiol. 13(5):775-86); celA (Parker, L.L., and Hall, B.G. 1990.Characterization and nucleotide sequence of the cryptic cel operon ofEscherichia coli K12. Genetics. 124(3):455-71); celB (Id.); celC (Id.);citA (Berlyn et al., “Linkage Map of Escherichia coli K-12, Edition 9,”Chapter 109 in: Escherichia coli and Salmonella typhimurium: Cellularand Molecular Biology, 2nd Ed., Neidhardt, Frederick C., Editor inChief, American Society for Microbiology, Washington, D.C., 1996, Volume2, pages 1715-1902, and references cited therein); citB (Id.); codB(Danielsen, S., et al. 1992. Characterization of the Escherichia colicodBA operon encoding cytosine permease and cytosine deaminase. Mol.Microbiol. 6(10):1335-44); cysA (Karbonowska, H., et al. 1977. Sulphatepermease of Escherichia coli K12. Acta. Biochim. Pol. 24(4):329-34);cysU (cysT) (Sirko, A., et al. 1995. Sulfate and thiosulfate transportin Escherichia coli K-12: evidence for a functional overlapping ofsulfate- and thiosulfate-binding proteins. J. Bacteriol.177(14):4134-6); cysW (Id.); dctA (Lo, T. C., and Bewick, M. A. 1978.The molecular mechanisms of dicarboxylic acid transport in Escherichiacoli K12. The role and orientation of the two membrane-bounddicarboxylate binding proteins. J. Biol. Chem. 10;253(21):7826-31); dctB(Id.); dcuA (genA) (Six, S., et al. 1994. Escherichia coli possesses twohomologous anaerobic C4-dicarboxylate membrane transporters (DcuA andDcuB) distinct from the aerobic dicarboxylate transport system (Dct). J.Bacteriol. 176(21):6470-8); dcuB (genF) (.); dgoT (Berlyn et al.,“Linkage Map of Escherichia coli K-12, Edition 9, ” Chapter 109 in:Escherichia coli and Salmonella typhimurium: Cellular and MolecularBiology, 2nd Ed., Neidhardt, Frederick C., Editor in Chief, AmericanSociety for Microbiology, Washington, D.C., 1996, Volume 2, pages1715-1902, and references cited therein); exuT (Nemoz, G., et al. 1976.Physiological and genetic regulation of the aldohexuronate transportsystem in Escherichia coli. J. Bacteriol. 127(2):706-18); fepD(Ozenberger, B. A., et al. 1987. Genetic organization of multiple fepgenes encoding ferric enterobactin transport functions in Escherichiacoli. J. Bacteriol. 169(8):3638-46); fepG (Chenault, S. S., and Earhart,C. F. 1991. Organization of genes encoding membrane proteins of theEscherichia coli ferrienterobactin permease. Mol. Microbiol.5(6):1405-13); fucP (prd) (Chen, Y. M. 1987. The organization of the fucregulon specifying L-fucose dissimilation in Escherichia coli K12 asdetermined by gene cloning. Mol. Gen. Genet. 210(2):331-7); glnP(Masters, P. S., and Hong, J. S. 1981. Genetics of the glutaminetransport system in Escherichia coli. J. Bacteriol. 147(3):805-19); glnQ(Nohno, T. 1986. Cloning and complete nucleotide sequence of theEscherichia coli glutamine permease operon (glnHPQ). Mol. Gen. Genet.205(2):260-9); glnR (Masters, P. S., and Hong, J. S. 1981. Genetics ofthe glutamine transport system in Escherichia coli. J. Bacteriol.147(3):805-19); glpT (Boos, W., et al. 1977. Purification and propertiesof a periplasmic protein related to sn-glycerol-3-phosphate transport inEscherichia coli. Eur. J. Biochem. 72(3):571-81); gltP (Deguchi, Y., etal. 1989. Molecular cloning of gltS and gltP, which encode glutamatecarriers of Escherichia coli. B. J. Bacteriol. 171(3):1314-9); gltS(Id.); gntR (Bachi, B., and Kornberg, H. L. 1975. Genes involved in theuptake and catabolism of gluconate by Escherichia coli. J. Gen.Microbiol. 90(2):321-35); gntS (Id.); gntT (gntM, usgA) (Id.); gntU(Tong, S. 1996. Cloning and molecular genetic characterization of theEscherichia coli gntR, gntK, and gntU genes of GntI, the main system forgluconate metabolism. J. Bacteriol. 178(11):3260-9); hisM (Berlyn etal., “Linkage Map of Escherichia coli K-12, Edition 9,” Chapter 109 in:Escherichia coli and Salmonella typhimurium: Cellular and MolecularBiology, 2nd Ed., Neidhardt, Frederick C., Editor in Chief, AmericanSociety for Microbiology, Washington, D.C., 1996, Volume 2, pages1715-1902, and references cited therein); hisP (Id.); hisQ (Id.); livG(hrbB, hrbC, hrbD) (Landick, R., et al. 1980. Regulation ofhigh-affinity leucine transport in Escherichia coli. J. Supramol.Struct. 14(4):527-37); livH (hrbB, hrbC, hrbD) (Id.); livJ (hrbB, hrbC,hrbD) (Id.); livK (hrbB, hrbC, hrbD) (Id.); livM (Id.); lldP (lctP)(Dong, J. M., et al. 1993. Three overlapping lct genes involved inL-lactate utilization by Escherichia coli. J. Bacteriol.175(20):6671-8); lysP (cadR) (Steffes, C., et al. 1992. The lysP geneencodes the lysine-specific permease. J. Bacteriol. 174(10):3242-9);malF (malB) (Bavoil, P., et al. 1980. Identification of a cytoplasmicmembrane-associated component of the maltose transport system ofEscherichia coli. J. Biol. Chem. 255(18):8366-9); malG (malB) (Dassa,E., and Hofnung, M. 1985. Sequence of gene malG in E. coli K12:homologies between integral membrane components from bindingprotein-dependent transport systems. EMBO J. 4(9):2287-93); malK (malB)(Id.); mglC (PMG, mglP) (Harayama, S. 1983. Characterization of the mgloperon of Escherichia coli by transposon mutagenesis and molecularcloning. J. Bacteriol. 153(1):408-15); nanT (Vimr, E. R., and Troy, F.A. 1985. Identification of an inducible catabolic system for sialicacids (nan) in Escherichia coli. J. Bacteriol. 164(2):845-53); nupC(cru) (Craig, J. E., et al. 1994. Cloning of the nupC gene ofEscherichia coli encoding a nucleoside transport system, andidentification of an adjacent insertion element, IS 186. Mol. Microbiol.11(6):1159-68); nupG (Westh Hansen, S.E., et al. 1987. Studies on thesequence and structure of the Escherichia coli K-12 nupG gene, encodinga nucleoside-transport system. Eur. J. Biochem. 168(2):385-91); panF(Vallari, D. S., and Rock, C. O. 1985. Isolation and characterizationEscherichia coli pantothenate permease (panF) mutants. J. Bacteriol.164(1): 136-42); potA (Kashiwagi, K., et al. 1993. Functions of potA andpotD proteins in spermidine-preferential uptake system in Escherichiacoli. J. Biol. Chem. 268(26):19358-63); potG (Pistocchi, R., et al.1993. Characteristics of the operon for a putrescine transport systemthat maps at 19 minutes on the Escherichia coli chromosome. J. Biol.Chem. 268(1): 146-52); potH (Id.); potI (Id.); proP (Wood, J. M., andZadworny, D. 1980. Amplification of the put genes and identification ofthe put gene products in Escherichia coli K12. Can. J. Biochem.58(10):787-96); proT (Id.); proV (proU) (Faatz, E., et al. 1988. Clonedstructural genes for the osmotically regulated binding-protein-dependentglycine betaine transport system (ProU) of Escherichia coli K-12. Mol.Microbiol. 2(2):265-79); proW (proU) (Id.); proX (proU) (Id.); pstA(R2pho, phoR2b, phoT) (Amemura, M., et al. 1985. Nucleotide sequence ofthe genes involved in phosphate transport and regulation of thephosphate regulon in Escherichia coli. J. Mol. Biol. 184(2):241-50);pstB (phoT) (Id.); pstC (phoW) (Rao, N. N., and Torriani, A. 1990.Molecular aspects of phosphate transport in Escherichia coli. Mol.Microbiol. 4(7): 1083-90); pstS (R2pho, nmpA, phoR2a, phoS) (Makino, K.,et al. 1988. Regulation of the phosphate regulon of Escherichia coli.Activation of pstS transcription by PhoB protein in vitro. J. Mol. Biol.203(1):85-95); purP (Burton, K. 1994. Adenine transport in Escherichiacoli. Proc. R. Soc. Lond. B. Biol. Sci. 255(1343):153-7); putP(Stalmach, M. E., et al. 1983. Two proline porters in Escherichia coliK-12. J. Bacteriol. 156(2):481-6); rbsA (rbsP, rbsT) (Iida, A., et al.1984. Molecular cloning and characterization of genes required forribose transport and utilization in Escherichia coli K-12. J. Bacteriol.158(2):674-82); rbsC (rbsP, rbsT) (Id.); rbsD (rbsP) (Id.); rhaT(Baldoma, L., et al. 1990. Cloning, mapping and gene productidentification of rhaT from Escherichia coli K12. FEMS Microbiol. Lett.60(1-2):103-7); sdaC (Shao, Z., et al. 1994. Sequencing andcharacterization of the sdaC gene and identification of the sdaCB operonin Escherichia coli K12. Eur. J. Biochem. 222(3):901-7); tnaB (trpP)(Sarsero, J. P., et al. 1991. A new family of integral membrane proteinsinvolved in transport of aromatic amino acids in Escherichia coli. J.Bacteriol. 173(10):3231-4); tyrR (Whipp, M. J., and Pittard, A. J. 1977.Regulation of aromatic amino acid transport systems in Escherichia coliK-12. J. Bacteriol. 132(2):453-61); ugpC (Schweizer, H., and Boos, W.1984. Characterization of the ugp region containing the genes for thephoB dependent sn-glycerol-3-phosphate transport system of Escherichiacoli. Mol. Gen. Genet. 197(1): 161-8); uhpT (Weston, L. A., and Kadner,R. J. 1987. Identification of uhp polypeptides and evidence for theirrole in exogenous induction of the sugar phosphate transport system ofEscherichia coli K-12. J. Bacteriol. 169(8):3546-55); and xylF (xylT)(Sumiya, M., et al. 1995. Molecular genetics of a receptor protein forD-xylose, encoded by the gene xylF, in Escherichia coli. ReceptorsChannels. 3(2):117-28).

[0372] II.C.6.b. Bacillus subtilis Genes

[0373] By way of non-limiting example, manipulation of the aapA gene orgene product from B. subtilis, or homologs of this gene or gene productfound in other members of the Prokaryotes, Eukaryotes, Archaebacteriaand/or organelles (e.g., mitochondria, chloroplasts, plastids and thelike) may be employed to increase the efficiency of gene expression andprotein production in parent cells prior to minicell formation and/or insegregated minicells (Sohenshein, A. L., J. A. Hoch, and R. Losick(eds.) 2002. Bacillus subtils and its closest relatives: from genes tocells. American Society for Microbiology, Washington D.C.).

[0374] In addition to aapA, other exemplary B. subtilis genes encodingfactors involved in the transport of inducers, inhibitors and othercompounds include, but are not limited to, amyC (Sekiguchi, J., et al.1975. Genes affecting the productivity of alpha-amylase in Bacillussubtilis. J. Bacteriol. 121(2):688-94); amyD (Id.); araE (Sa-Nogueira,I., and Mota, L. J. 1997. Negative regulation of L-arabinose metabolismin Bacillus subtilis: characterization of the araR (araC) gene. J.Bacteriol. 179(5): 1598-608); araN (Sa-Nogueira, I., et al. 1997. TheBacillus subtilis L-arabinose (ara) operon: nucleotide sequence, geneticorganization and expression. Microbiology. 143 (Pt 3):957-69); araP(Id.); araQ (Id.); csbC (Akbar, S., et al. 1999. Two genes from Bacillussubtilis under the sole control of the general stress transcriptionfactor sigmaB. Microbiology. 145 (Pt 5):1069-78); cysP (Mansilla, M. C.,and de Mendoza, D. 2000. The Bacillus subtilis cysP gene encodes a novelsulphate permease related to the inorganic phosphate transporter (Pit)family. Microbiology. 146 ( Pt 4):815-21); dctB (Sohenshein, A. L., J.A. Hoch, and R. Losick (eds.) 2002. Bacillus subtils and its closestrelatives: from genes to cells. American Society for Microbiology,Washington D.C.); exuT (Rivolta, C., et al. 1998. A 35.7 kb DNA fragmentfrom the Bacillus subtilis chromosome containing a putative 12.3 kboperon involved in hexuronate catabolism and a perfectly symmetricalhypothetical catabolite-responsive element. Microbiology. 144 ( Pt4):877-84); gabP (Ferson, A. E., et al. 1996. Expression of the Bacillussubtilis gabP gene is regulated independently in response to nitrogenand amino acid availability. Mol. Microbiol. 22(4):693-701); gamP(Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillussubtils and its closest relatives: from genes to cells. American Societyfor Microbiology, Washington D.C.); glcP (Paulsen, I. T., et al. 1998.Characterization of glucose-specific catabolite repression-resistantmutants of Bacillus subtilis: identification of a novel hexose:H+symporter. J. Bacteriol. 180(3):498-504); glcU (Sohenshein, A. L., J. A.Hoch, and R. Losick (eds.) 2002. Bacillus subtils and its closestrelatives: from genes to cells. American Society for Microbiology,Washington D.C.); glnH (Id.); glnM (Id); glnP (Sohenshein, A. L., J. A.Hoch, and R. Losick (eds.) 2002. Bacillus subtils and its closestrelatives: from genes to cells. American Society for Microbiology,Washington D.C.); glnQ (Id.); glpT (Nilsson, R. P., et al. 1994. TheglpT and glpQ genes of the glycerol regulon in Bacillus subtilis.Microbiology. 140 (Pt 4):723-30); gltP (Tolner, B., et al. 1995.Characterization of the proton/glutamate symport protein of Bacillussubtilis and its functional expression in Escherichia coli. J.Bacteriol. 177(10):2863-9); gltT (Tolner, B., et al. 1995.Characterization of the proton/glutamate symport protein of Bacillussubtilis and its functional expression in Escherichia coli. J.Bacteriol. 177(10):2863-9); gntP (Reizer, A., et al. Analysis of thegluconate (gnt) operon of Bacillus subtilis. Mol. Microbiol.5(5):1081-9); gutP (Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.)2002. Bacillus subtils and its closest relatives: from genes to cells.American Society for Microbiology, Washington D.C.); hutM (Oda, M., etal. 1988. Cloning and nucleotide sequences of histidase and regulatorygenes in the Bacillus subtilis hut operon and positive regulation of theoperon. J. Bacteriol. 170(7):3199-205); iolF (Sohenshein, A. L., J. A.Hoch, and R. Losick (eds.) 2002. Bacillus subtils and its closestrelatives: from genes to cells. American Society for Microbiology,Washington D.C.); kdgT (Pujic, P., et al. 1998. The kdgRKAT operon ofBacillus subtilis: detection of the transcript and regulation by thekdgR and ccpA genes. Microbiology. 144 (Pt 11): 3111-8); lctP (Cruz,Ramos H., et al. 2000. Fermentative metabolism of Bacillus subtilis:physiology and regulation of gene expression. J. Bacteriol.182(11):3072-80); maeN (Ito, M., et al. 2000. Effects of nonpolarmutations in each of the seven Bacillus subtilis mrp genes suggestcomplex interactions among the gene products in support of Na(+) andalkali but not cholate resistance. J. Bacteriol. 182(20):5663-70); malP(Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillussubtilis and its closest relatives: from genes to cells. AmericanSociety for Microbiology, Washington D.C.); manP (Id.); mleN (Id.); nasA(Ogawa, K., et al. 1995. The nasB operon and nasA gene are required fornitrate/nitrite assimilation in Bacillus subtili. J. Bacteriol.177(5):1409-13); nupC (Sohenshein, A. L., J. A. Hoch, and R. Losick(eds.) 2002. Bacillus subtils and its closest relatives: from genes tocells. American Society for Microbiology, Washington D.C.); opuAB(Kempf, B., et al. 1997. Lipoprotein from the osmoregulated ABCtransport system OpuA of Bacillus subtilis: purification of the glycinebetaine binding protein and characterization of a functional lipidlessmutant. J. Bacteriol. 179(20):6213-20); opuBA (Sohenshein, A. L., J. A.Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and its closestrelatives: from genes to cells. American Society for Microbiology,Washington D.C.); pbuG (Saxild, H. H., et al. 2001. Definition of theBacillus subtilis PurR operator using genetic and bioinformatic toolsand expansion of the PurR regulon with glyA, guaC, pbuG, xpt-pbuX,yqhZ-folD, and pbuO. J. Bacteriol. 183(21):6175-83); pbuX (Saxild, H.H., et al. 2001. Definition of the Bacillus subtilis PurR operator usinggenetic and bioinformatic tools and expansion of the PurR regulon withglyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO. J. Bacteriol.183(21):6175-83); pstC (Takemaru, K., et al. 1996. A Bacillus subtilisgene cluster similar to the Escherichia coli phosphate-specifictransport (pst) operon: evidence for a tandemly arranged pstB gene.Microbiology. 142 ( Pt 8):2017-20); pstS (Qi, Y., et al. 1997. The pstoperon of Bacillus subtilis has a phosphate-regulated promoter and isinvolved in phosphate transport but not in regulation of the phoregulon. J. Bacteriol. 179(8):2534-9); pucJ (Schultz, A. C., et al.2001. Functional analysis of 14 genes that constitute the purinecatabolic pathway in Bacillus subtilis and evidence for a novel reguloncontrolled by the PucR transcription activator. J. Bacteriol.183(11):3293-302); pucK (Schultz, A. C., et. al. 2001. Functionalanalysis of 14 genes that constitute the purine catabolic pathway inBacillus subtilis and evidence for a novel regulon controlled by thePucR transcription activator. J. Bacteriol. 183(11):3293-302); pyrP(Turner, R. J., et al. 1994. Regulation of the Bacillus subtilispyrimidine biosynthetic (pyr) gene cluster by an autogenoustranscriptional attenuation mechanism. J. Bacteriol. 176(12):3708-22);rbsB (Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillussubtils and its closest relatives: from genes to cells. American Societyfor Microbiology, Washington D.C.); rbsC (Sohenshein, A. L., J. A. Hoch,and R. Losick (eds.) 2002. Bacillus subtils and its closest relatives:from genes to cells. American Society for Microbiology, WashingtonD.C.); rbsD (Id.); rocC (Gardan, R., et al. 1995. Expression of therocDEF operon involved in arginine catabolism in Bacillus subtilis. J.Mol. Biol. 23;249(5):843-56); rocE (Gardan, R., et al. 1995. Expressionof the rocDEF operon involved in arginine catabolism in Bacillussubtilis. J. Mol. Biol. 23;249(5):843-56); ssuA (Coppee, J. Y., et al.2001. Sulfur-limitation-regulated proteins in Bacillus subtilis: atwo-dimensional gel electrophoresis study. Microbiology. 147(Pt6):1631-40); ssuB (van der Ploeg, J. R., et al. 1998. Bacillus subtilsgenes for the utilization of sulfur from aliphatic sulfonates.Microbiology. 144 ( Pt 9):2555-61); ssuC (van der Ploeg, J. R., et al.1998. Bacillus subtils genes for the utilization of sulfur fromaliphatic sulfonates. Microbiology. 144 ( Pt 9):2555-61); treP(Yamamoto, H., et al. 1996. Cloning and sequencing of a 40.6 kb segmentin the 73 degrees-76 degrees region of the Bacillus subtilis chromosomecontaining genes for trehalose metabolism and acetoin utilization.Microbiology. 142 ( Pt 11):3057-65); xynP (Sohenshein, A. L., J. A.Hoch, and R. Losick (eds.) 2002. Bacillus subtils and its closestrelatives: from genes to cells. American Society for Microbiology,Washington D.C.); ybaR (Id.); ybgF (Id.); ybgH (Id.); ycbE (Id.); ycgO(Id..); yckI (Id.); yckJ (Id.); yckK (Id.); ydgF (Id.); yecA (Borriss,R., et al. 1996. The 52 degrees-55 degrees segment of the Bacillussubtilis chromosome: a region devoted to purine uptake and metabolism,and containing the genes cotA, gabP and guaA and the pur gene clusterwithin a 34960 bp nucleotide sequence. Microbiology. 142 (Pt11):3027-31); yesP (Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.)2002. Bacillus subtils and its closest relatives: from genes to cells.American Society for Microbiology, Washington D.C.); yesQ (Id.): yflS(Id.); yhcL (Id.); yhjB (Id.); yjkB (Id.); ykbA (Id.); yoaB (Id.); yocN(Id.); yodF (Id.); yojA (Id.); yqiY (Id.); ytlD (Id.); ytlP (Id.); ytmL(Id.); ytmM (Id.); ytnA (Id.); yurM (Id.); yurN (Id.); yvbW (Id.); yvdH(Id.); yvdI (Id.); yveA (Pereira, Y., et al. 2001. The yveB gene,Encoding endolevanase LevB, is part of the sacB-yveB-yveA levansucrasetricistronic operon in Bacillus subtilis. Microbiology. 147(Pt12):3413-9); yvfH (Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.)2002. Bacillus subtils and its closest relatives: from genes to cells.American Society for Microbiology, Washington D.C.); yvfL (Id.); yvfM(Id.); yvgM (Id.); yvrO (Id.); yvsH (Id.); ywbF (Id.); ywcJ (Id.); ywoD(Id.); ywoE (Id.); yxeN (Id.); and yxeR (Id.).

[0375] II.C.7. Catabolite Repression

[0376] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inminicells. By way of non-limiting example, these techniques may includeutilization and/or modification of factors and systems involved in thesynthesis, degradation or transport of catabolites that modulate thegenetic expression of a preselected protein. Such manipulations mayresult in increased or decreased production, and/or changes in theintramolecular and intermolecular functions, of a protein in a minicellor its parent cell; in the latter instance, the protein may be one thatis desirably retained in segregated minicells.

[0377] By way of non-limiting example, it is known in the art to usepromoters from the trp, cst-1, and llp operons of E. coli, which areinduced by, respectively, reduced tryptophan levels, glucose starvation,and lactose. Manipulation of the catabolites tryptophan, glucose andlactose, respectively, will influence the degree of expression of genesoperably linked to these promoters. (Makrides, Savvas C., Strategies forAchieving High-Level Expression of Genes in Escherichia coli.Microbiological Reviews. 1996. 60:512-538.)

[0378] As another non-limiting example, expression elements from the E.coli L-arabinose (ara) operon are used in expression systems. AraC is aprotein that acts as a repressor of ara genes in the absence ofarabinose, and also as an activator of ara genes when arabinose ispresent. Induction of ara genes also involves cAMP, which modulates theactivity of CRP (cAMP receptor protein), which in turn is required forfull induction of ara genes (Schleif, Robert, Regulation of theL-arabinose operon of Escherichia coli. 2000. TIG 16:559-564. Thus,maximum expression from an ara-based expression system is achieved byadding cAMP and arabinose to host cells, and optimizing the expressionof CRP in hostcells.

[0379] As one example, manipulation of the acpS gene or gene product ofE. coli (Pollacco M. L., and J. E. Cronan Jr. 1981. A mutant ofEscherichia coli conditionally defective in the synthesis of holo-[acylcarrier protein]. J. Biol.Chem. 256:5750-5754); or homologs of this geneor its gene product found in other prokaryotes, eukaryotes,archaebacteria or organelles (mitochondria, chloroplasts, plastids andthe like) may be employed to increase the efficiency of gene expressionand protein production in parent cells prior to minicell formationand/or in segregated minicells.

[0380] In addition to acpS, other exemplary E. coli genes include theb2383 gene (Berlyn et al., “Linkage Map of Escherichia coli K-12,Edition 9,” Chapter 109 in: Escherichia coli and Salmonella typhimurium:Cellular and Molecular Biology, 2nd Ed., Neidhardt, Frederick C., Editorin Chief, American Society for Microbiology, Washington, D.C., 1996,Volume 2, pages 1715-1902, and references cited therein. b2387 gene; thecelA gene (Parker L. L., and B. G. Hall. 1990. Characterization andnucleotide sequence of the cryptic cel operon of Escherichia coli K12.Genetics. 124:455-471); the celB gene (Cole S. T., and B. Saint-Joanis,and A. P. Pugsley. 1985. Molecular characterisation of the colicin E2operon and identification of its products. Mol Gen Genet. 198:465-472);the celC gene (Parker L. L., and B. G. Hall. 1990. Characterization andnucleotide sequence of the cryptic cel operon of Escherichia coli K12.Genetics. 124:455-471); the cmtB gene (Ezhova N. M., Zaikina, N. A,Shataeva,L. K., Dubinina, N. I., Ovechkina, T. P. and J. V. Kopylova.[Sorption properties of carboxyl cation exchangers with a bacteriostaticeffect]. 1980. Prikl Bioikhim Mikrobiol. 16:395-398); the creB gene(Berlyn et al., −Linkage Map of Escherichia coli K-12, Edition 9”Chapter 109 in: Escherichia coli and Salmonella typhimurium: Cellularand Molecular Biology, 2nd Ed., Neidhardt, Frederick C., Editor inChief, American Society for Microbiology, Washington, D.C., 1996, Volume2, pages 1715-1902, and references cited therein; the creC gene (WannerB. L. Gene regulation by phosphate in enteric bacteria. 1993. J CellBiochem. 51:47-54); the crp gene (Sabourn D., and J. Beckwith. Deletionof the Escherichia coli crp gene. 1975. J Bacteriology. 122:338-340);the crr (gsr, iex, tgs, treD) gene (Jones-Mortimer M. C., and H. L.Kornberg, and r. Maltby, and P. D. Watts. Role of the crr-gene inglucose uptake by Escherichia coli. 1977. FEBS Lett. 74:17-19); the cyagene (Bachi B., and H. L. Kornberg. Utilization of gluconate byEscherichia coli. A role of adenosine 3′:5′-cyclic monophosphate in theinduction of gluconate catabolism. 1975. Biochem J. 150:123-128); thefruA gene (Prior T. I., and H. L. Kornberg. Nucleotide sequence of fruA,the gene specifying enzyme Iifru of the phosphoenopyruvate-dependentsugar phosphotranssferase system in Escherichia coli K12. 1988. J GenMicrobiol. 134:2757-2768); the fruB gene (Bol'shakova T. N. and R. S.Erlagaeva, and Dobrynina Oiu, and V. N. Gershanovich. [Mutation fruB inthe fructose regulon affeting beta-galactosidase synthesis and adenylatecyclase activity of E. coli K12]. 1988. Mol Gen Mikrobiol virusol.3:33-39); the fruR gene (Jahreis K., and P. W. Postma, and J. W.Lengeler. Nucleotide sequence of the ilvH-frR gene region of Escherichiacoli K12 and Salmonella typhimurium LT2. 1991. Mol Gen Genet.226:332-336); the frvA gene (Berlyn et al., “Linkage Map of Escherichiacoli K-12, Edition 9,” Chapter 109 in: Escherichia coli and Salmonellatyphimurium: Cellular and Molecular Biology, 2nd Ed., Neidhardt,Frederick C., Editor in Chief, American Society for Microbiology,Washington, D.C., 1996, Volume 2, pages 1715-1902, and references citedtherein); the frwB gene (Id.); the fruD gene (Id.); the gatB gene(Nobelmann B., and J. W. Lengeler. Molecular analysis of the gat genesfrom Escherichia coli and of their roles in galactitol transport andmetabolism. 1996. J Bacteriol. 178:6790-6795); the gatC gene (Id.); themalX gene (Reidel J., W. Boos. The malX malY operon of Escherichia coliencodes a novel enzyme II of the photophotransferase system recognizingglucose and maltose and an enzyme abolishing the endogenous induction ofthe maltose system. 1991. J Bacteriol. 173:4862-4876); the manX (gptB,mpt, ptsL, ptsM, ptsX, manIII) gene (Plumbridge J., and A. Kolb. CAP andNag repressor binding to the regulatory regions of the nagE-B and manXgenes of Escherichia coli. 1991. J Mol Biol. 217:661-679); the manY(pel, ptsM, ptsP, manPII) gene (Henderson P. J., and R. A. Giddens, andM. C. Jones-Mortimer. Transport of galactose, glucose and theirmolecular analogues by Escherichia coli K12. 1977. Biochem J.162:309-320); the manZ (gptB, mpt, ptsM, ptsX) gene (Williams N., and D.K. Fox, and C. Shea and S. Roseman. Pel, the protein that permits lambdaDNA penetration of Escherichia coli, is encoded by a gene in ptsM and isrequired for mannose utilization by the phosphotransferase system. 1986.Proc Natl Acad Sci USA. 83:8934-8938); the mtlA gene (Lengeler J.Mutations affecting transport of the hexitols D-mannitol, D-glucitol,and galactitol in Escherichia coli K-12: isolation and mapping. 1975. JBacteriol. 124:26-38.); the nagE (pstN) gene (Rogers M. J., and T. Ohgi,and J. Plumbridge, and D. Soll. Nucleotide sequences of the Escherichiacoli nagE and nagB genes: the structural genes for theN-acetylglucosamine transport protein of the bacterialphosphoenolpyruvate: sugar phosphotransferase system and forglucosamine-6-phosphate deaminase. 1988. Gene. 62:197-207); the pstAgene (Cox G. B., H. Rosenberg, and J. A. Downie, and S. Silver. Geneticanalysis of mutants affected in the Pst inorganic phosphate transportsystem. 1981. J Bacteriol. 148:1-9); the pstB (gutB) gene (Id.); thepstG gene (Cox G. B., H. Rosenberg, and J. A. Downie, and S. Silver.Genetic analysis of mutants affected in the Pst inorganic phosphatetransport system. 1981. J Bacteriol. 148:1-9); the pstH gene (Id.); thepstI gene (Id.); the pstN gene (Id.); the pstO gene (Id.); the ptxA(yifU) gene (Berlyn et al., “Linkage Map of Escherichia coli K-12,Edition 9,” Chapter 109 in: Escherichia coli and Salmonella typhimurium:Cellular and Molecular Biology, 2nd Ed., Neidhardt, Frederick C., Editorin Chief, American Society for Microbiology, Washington, D.C., 1996,Volume 2, pages 1715-1902, and references cited therein); the sgcA(yjhL) gene (Id.); the sgcC (yjhN) gene (Id.); the treB gene (Boos W.,U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma. Trehalosetransport and metabolism in Escherichia coli. 1990. J. Bacteriol.172:3450-3461); the usg gene (Arps P. J., and M. E. Winkler M E.Structural analysis of the Escherichia coli K-12 hisT operon by using akanamycin resistance cassette. 1987. J Bacteriol. 169:1061-1070); thewcaD gene (Mao Y., and M. P. Doyle, and J. Chen. Insertion mutagenisisof wca reduces acide and heat tolerance of enterohemorrhagic Escherichiacoli O157:H7. 2001. J Bacteriol. 183:3811-3815); the yadI gene (Berlynet al., “Linkage Map of Escherichia coli K-12, Edition 9,” Chapter 109in: Escherichia coli and Salmonella typhimurium: Cellular and MolecularBiology, 2nd Ed., Neidhardt, Frederick C., Editor in Chief, AmericanSociety for Microbiology, Washington, D.C., 1996, Volume 2, pages1715-1902, and references cited therein); and the ycgC gene (GutknechtR., and R. Beutler, and L. F. Garcia-Alles, and U. Baumann, and B. Erni.The dihydroxyacetone kinase of Escherichia coli utilizes aphosphoprotein instead of ATP as phosphoryl donor. 2001. EMBO J.20:2480-2486).

[0381] II. C.8. General Deletions and Modifications

[0382] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inminicells. By way of non-limiting example, these techniques may includemodification or deletion of endogenous gene(s) from which theirrespective gene product decreases the induction and expressionefficiency of a desired protein in the parent cell prior to minicellformation and/or the segregated minicell. By way of non-limitingexample, these protein components may be the enzymes that degradechemical inducers of expression, proteins that have a dominant negativeaffect upon a positive regulatory elements, proteins that haveproteolytic activity against the protein to be expressed, proteins thathave a negative affect against a chaperone that is required for properactivity of the expressed protein, and/or this protein may have apositive effect upon a protein that either degrades or prevents theproper function of the expressed protein. These gene products thatrequire deletion or modification for optimal protein expression and/orfunction may also be antisense nucleic acids that have a negative affectupon gene expression.

[0383] II.C.9. Cytoplasmic Redox State

[0384] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and functional proteinproduction in minicells. By way of non-limiting example, thesetechniques may include modification of endogenous and/or exogenousprotein components that alter the redox state of the parental cellcytoplasm prior to minicell formation and/or the segregated minicellcytoplasm. By way of non-limiting example, this protein component may bethe product of the trxA, grx, dsbA, dsbB, and/or dsbc genes from E. colior homologs of this gene or gene product found in other members of theEubacteria, Eucarya or Archae (Mark et al., Genetic mapping of trxA, agene affecting thioredoxin in Escherichia coli K12, Mol Gen Genet.155:145-152, 1977; (Russel et al., Thioredoxin or glutaredoxin inEscherichia coli is essential for sulfate reduction but not fordeoxyribonucleotide synthesis, J Bacteriol. 172:1923-1929, 1990);Akiyama et al., In vitro catalysis of oxidative folding ofdisulfide-bonded proteins by the Escherichia coli dsbA (ppfA) geneproduct, J Biol Chem. 267:22440-22445, 1992); (Whitney et al., TheDsbA-DsbB system affects the formation of disulfide bonds in periplasmicbut not in intramembraneous protein domains, FEBS Lett. 332:49-51,1993); (Shevchik et al., Characterization of DsbC, a periplasmic proteinof Erwinia chrysanthemi and Escherichia coli with disulfide isomeraseactivity, EMB J. 13:2007-2012, 1994). These applications may, but arenot limited to increased or decreased production, increased or decreasedintramolecular TrxA activity, increased or decreased physiologicalfunction of the above-mentioned gene products. By way of non-limitingexample, increased production of gene product (gene expression) mayoccur through increased gene dosage (increased copy number of a givengene under the control of the native or artificial promotor where thisgene may be on a plasmid or in more than one copy on the chromosome),modification of the native regulatory elements, including, but notlimited to the promotor or operator region(s) of DNA, or ribosomalbinding sites on RNA, relevant repressors/silencers, relevantactivators/inhancers, or relevant antisense nucleic acid or nucleic acidanalog, cloning on a plasmid under the control of the native orartificial promotor, and increased or decreased production of native orartificial promotor regulatory elements) controlling production of thegene. By way of non-limiting example, decreased gene expressionproduction may occur through modification of the native regulatoryelements, including, but not limited to the promotor or operatorregion(s) of DNA, or ribosomal binding sites on RNA, relevantrepressors/silencers, relevant activators/inhancers, or relevantantisense nucleic acid or nucleic acid analog, through cloning on aplasmid under the control of the native regulatory region containingmutations or an artificial promotor, either or both of which resultingin decrease gene expression, and through increased or decreasedproduction of native or artificial promotor regulatory element(s)controlling gene expression. By definition, intramolecular activityrefers to the enzymatic function, structure-dependent function, e.g. thecapacity off a gene product to interact in a protein-protein,protein-nucleic acid, or protein-lipid complex, and/or carrier function,e.g. the capacity to bind, either covalently or non-covalently smallorganic or inorganic molecules, protein(s) carbohydrate(s), fattyacid(s), lipid(s), and nucleic acid(s). By way of non-limiting example,alteration of intramolecular activity may be accomplished by mutation ofthe gene, in vivo or in vitro chemical modification of the gene product,inhibitor molecules against the function of the gene product, e.g.competitive, non-competitive, or uncompetitive enzymatic inhibitors,inhibitors that prevent protein-protein, protein-nucleic acid, orprotein-lipid interactions, e.g. expression or introduction ofdominant-negative or dominant-positive or other protein fragment(s), orother carbohydrate(s), fatty acid(s), lipid(s), and nucleic acid(s) thatmay act directly or allosterically upon the gene product, and/ormodification of protein, carbohydrate, fatty acid, lipid, or nucleicacid moieties that modify the gene or gene product to create thefunctional protein. By definition, physiological function refers to theeffects resulting from an intramolecular interaction between the geneproduct and other protein, carbohydrate, fatty acid, lipid, nucleicacid, or other molecule(s) in or on the cell or the action of a productor products resulting from such an interaction.

[0385] By way of non-limiting example, physiological function may be theact or result of intermolecular phosphorylation, biotinylation,methylation, acylation, glycosylation, and/or other signaling event;this function may be the result of protein-protein, protein-nucleicacid, or protein-lipid interaction resulting in a functional moiety;this function may be to interact with the membrane to recruit othermolecules to this compartment of the cell; this function may be toregulate the transcription and/or translation of trxA, other protein, ornucleic acid; and this function may be to stimulate the function ofanother process that is not yet described or understood.

[0386] II.C.10. Transcriptional Terminators

[0387] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inparental cell cytoplasm prior to minicell formation and/or thesegregated minicell cytoplasm. By way of non-limiting example, thesetechniques may include modification of terminator regions of DNAtemplates or RNA transcripts so that transcription and/or translation ofthese nucleic acid regions will terminate at greater efficiency. By wayof non-limiting example, these techniques may include stem-loopstructures, consecutive translational terminators, polyadenylationsequences, or increasing the efficiency of rho-dependent termination.Stem loop structures may include, but are not limited to, invertedrepeats containing any combination of deoxyribonucleic acid orribonucleic acid molecule, more than one such inverted repeat, orvariable inverted repeats such that the rate oftranscriptional/translational termination may be moderated dependent onnucleic acid and/or amino acid concentration, e.g. the mechanism ofregulatory attenuation (Oxdender et al., Attenuation in the Escherichiacoli tryptophan operon: role of RNA secondary structure involving thetryptophan codon region, Proc. Natl. Acad. Sci. 76:5524-5528, 1979). Seealso, Yager and von Hippel, “Transcript Elongation and Termination in e.Col. And Landick and Yanofsky, “Transcriptional Attenuation,” Chapters76 and 77, respectively in: Escherichia Coli and Salmonella Typhimurium:Cellular and Molecular Biology, Neidhardt, Frederick C., Editor inChief, American Society for Microbiology, Washington, D.C., 1987, Volume1, pages 1241-1275 and 1276-1301, respectively, and references citedtherein.

[0388] II.C.11. Ribosomal Targeting

[0389] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inparental cell cytoplasm prior to minicell formation and/or thesegregated minicell cytoplasm. By way of non-limiting example, thesetechniques may include modifications of endogenous and/or exogenousribosomal components such that ribosomes enter the minicell segregateswith higher efficiency. By way of non-limiting example, these techniquesmay include increasing the copy number of ribosomal binding sites onplasmid or like structure to recruit more ribosomal components orincrease the synthesis of ribosomal subunits prior to segregation (Mawnet al., Depletion of free 30S ribosomal subunits in Escherichia coli byexpression of RNA containing Shine-Dalgarno-like sequences, J.Bacteriol. 184:494-502, 2002). This construct may also include the useof plasmid expressed translation initiation factors to assist ribosomalsegregation (Celano et al., Interaction of Escherichia colitranslation-initiation factor IF-1 with ribosomes, Eur. J. Biochem.178:351-355 1988). See also Hoopes and McClure, “Strategies inRegulation of Transcription Initiation,” Chapter 75 in: Escherichia Coliand Salmonella Typhimurium: Cellular and Molecular Biology, Neidhardt,Frederick C., Editor in Chief, American Society for Microbiology,Washington, D.C., 1987, Volume 2, pages 1231-1240, and references citedtherein.

[0390] II.C. 12. Proteases

[0391] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inminicells. By way of non-limiting example, these techniques may includeutilization and/or modification of endogenous and/or exogenousproteases. Such manipulations may result in increased or decreasedproduction, and/or changes in the intramolecular and intermolecularfunctions, of a protein in a minicell or its parent cell; in the latterinstance, the protein may be one that is desirably retained insegregated minicells.

[0392] The production or activity of a desired protein gene product maybe increased by decreasing the level and/or activity of a protease thatacts upon the desired protein. The production or activity of a desiredprotein gene product may be increased by increasing the level and/oractivity of a protease that acts upon a protein that inhibits theproduction or function of the desired protein.

[0393] The production or activity of a desired nucleic acid gene productmay be increased be increasing the level and/or activity of a proteasethat acts upon a protein that that inhibits the production or functionof the nucleic acid gene product. The production or activity of adesired nucleic acid gene product may be increased by decreasing thelevel and/or activity of a protease that acts upon a protein thatstimulates or enhances the production or function of the desired nucleicacid gene product.

[0394] As one example, manipulation of the alpA gene or gene productfrom E. coli (Kirby J. E., and J. E. Trempy, and S. Gottesman. Excisionof a P4-like cryptic prophage leads to Alp protease expression inEscherichia coli. 1994. J Bacteriol. 176:2068-2081), or homologs of thisgene or gene product found in other members of the Prokaryotes,Eukaryotes or Archaebacteria, may be employed to increase the efficiencyof gene expression and protein production in parent cells prior tominicell formation and/or segregated minicells postpartum.

[0395] In addition to alpA, other exemplary E. coli genes and geneproducts include the clpA gene and gene product from E. coli (KatayamaY., and S. Gottesman, and J. Pumphrey, and S. Rudikoff, and W. P. Clark,and M. R. Maurizi. The two-component, ATP-dependent Clp protease ofEscherichia coli. Purification, cloning, and mutational analysis of theATP-binding component. 1988, J Biol Chem. 263-15226-15236); the clpBgene product from E. coli (Kitagawa M., and C. Wada, and S. Yoshioka,and T. Yura. Expression of ClpB, an analog of the ATP-dependent proteasregulatory subunit in Escherichia coli, is controlled by a heat shocksigma factor (sigma 32). J Bacteriol. 173:42474253); the clpC geneproduct from E. coli (Msadek T., and F. Kunst, and G. Rapoport. MecB ofBacillus subtilis, a member of the ClpC ATPase family, is a pleiotropicregulator controlling competence gene expression and growth at hightemperature. 1994. Proc Natl Acad Sci USA 91:5788-5792); the clpP geneproduct from E. coli (Maurizi M. R., and W. P. Clark, and Y. Katayama,and S. Rudikoff, and J. Pumphrey, and B. Bowers, and S. Gottesman.Sequence and structure of ClpP, the proteolytic component of theATP-dependent Clp protease of Escherichia coli. 1990. J biol Chem.265:12536-12545); the clpX gene product from E. coli (Gottesman S., andW. P. Clark, and V. de Crecy-Lagard, and M. R. Maurizi. ClpX, analternative subunit for the ATP-dependent Clp protease of Escherichiacoli. Sequence and in vivo activities. 1993. J Biol Chem.268:22618-22626); the clpY gene product from E. coli (Missiakas D., andF. Schwager, J. M. Betton, and C. Georgopoulos, S. Raina. Identificationand characterization of HsIV HsIU (ClpQ ClpY) proteins involved inoverall proteolysis of misfolded proteins in Escherichia coli. 1996.EMBO J. 15:6899-6909); the dcp gene product from E. coli (Becker S., andPlapp R. Location of the dcp gene on the physical map of Escherichiacoli. 1992. J Bacteriol. 174:1698-1699); the degP (htrA) gene productfrom E. coli (Lipinska B., and M. Zylicz, and C. Georgopoulos. The HtrA(DegP) protein, essential for Escherichia coli survival at hightemperatures, is an endopeptidase. 1990. J Bacteriol. 172:1791-1797);the ggt gene product from E. coli (Finidori J., and Y. Laperche, and R.Haguenauer-Tsapis, and R. Barouki, and G. Guellaen, and J. Hanoune. Invitro biosynthesis and membrane insertion of gamma-glutamyltranspeptidase. 1984. J Biol Chem. 259:4687-4690); the hfl gene productfrom E. coli (Cheng H. H., and H. Echols. A class of Escherichia coliproteins controlled by the hflA locus. 1987. J Mol Biol. 196:737-740);the hflB gene product from E. coli (Banuett F., and M. A. Hoyt, and L.McFarlane, and H. Echols, and I. Herskowitz. HflB, a new Escherichiacoli locus regulating lysogeny and the level of bacteriophage lambda c11protein. 1986. J Mol Biol. 187:213-224); the hflC gene product from E.coli (Noble J. A., and M. A. Innis, and E. V. Koonin, and K. E. Rudd,and F. Banuett, and I. Herskowitz, The Escherichia coli hflA locusencodes a putative GTP-binding protein and two membrane proteins, one ofwhich contains a protease-like domain. 1993. Proc Natl Acad Sci U S A.90:10866-10870); the hflK gene product from E. coli (Id.); the hftX geneproduct from E. coli (Noble J. A., and M. A. Innis, and E. V. Koonin,and K. E. Rudd, and F. Banuett, and I. Hertzskowitz. The Escherichiacoli hflA locus encodes a putative GTP-binding protein and two membraneproteins, one of which contains a protease-like domain. 1993. Proc NatlAcad Sci U S A. 90:10866-10870); the hopD gene product from E. coli(Whitchurch C. B., and J. S. Mattick Escherichia coli contains a set ofgenes homologous to those involved in protein secretion, DNA uptake andthe assembly of type-4 fimbriae in other bacteria. 1994. Gene.150:9-15); the htrA gene product from E. coli (Lipinska B., and S.Sharma, and C. Georgopoulos. Sequence analysis and regulation of thehtrA gene of Escherichia coli: a sigma 32-independent mechanism ofheat-inducible transcription. 1988. Nucleicc Acids Res. 16:10053-10067);the hycI gene product from E. coli (Rossmann R., and T. Maier, and F.Lottspeich, and A. Bock. Characterisation of a proteas from Escherichiacoli involved in hydrogenase maturation. 1995. Eur J Biochem.227:545-550); the iap gene product from E. coli (Nakata A., and M.Yamaguchi, and K. Isutani, and M. Amemura. Escherichia coli mutantsdeficient in the production of alkaline phosphatase isoszymes. 1978. JBacteriol. 134:287-294); the lep gene product from E. coli (Silver P.,and W. Wickner. Genetic mapping of the Escherichia coli leader (signal)peptidase gene (lep): a new approach for determining the map position ofa cloned gene. 1983. J Bacteriol. 54:659-572); the lon gene product fromE. coli (Donch J., and J. Greenberg. Genetic analysis of Ion mutants ofstrain K-12 of Escherichia coli. 1968. Mol Gen Genet. 103:105-115); thelsp gene product from E. coli (Regue M., and J. Remenick, and M.tokunaga, and G. A. Mackie, and H. C. Wu. Mapping of the lipoproteinsignal peptidase gene (lsp). 1984. J Bacteriol. 1984 158:632-635); theompT gene product from E. coli (Akiyama Y., and K. SecY protein, amembrane-embedded secretion factor of E. coli, is cleaved by the ompTproteas in vitro. 1990. Biochem Biophys Res Commun. 167:711-715); theopdA gene product from E. coli (Conllin C. A., and C. G. Miller.Location of the prlC (opdA) gene on the physical map of Escherichiacoli. 1993. J Bacteriiol. 175:5731-5732); the orfX gene product from E.coli (Berlyn, M. K. B., et al. 1996. Linkage map of Escherichia coliK-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C.C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.Schaechter, and H. E. Umbarger (eds.). Escherichia coli and Salmonellatyphimurium: Cellular and Molecular Biology, 2^(nd) ed. American Societyfor Microbiology, Washington D.C.); the pepA gene product from E. coli(Stirling C. J., and S. D. Colloms, and J. F. Collins, and G. Szatmari,and D. J. Sherratt. XerB, an Escherichia coli gene required for plasmidColE1 site-specific recombination, is identical to pepA, encodingaminopeptidaseA, a protein with substantial similarity to bovine lensleucine aminopeptidase. 1989. EMBO J. 8:1623-1627); the pepD geneproduct from E. coli (Henrich B., and U. Schroeder, and R. W. Frank, andR. Plapp. Accurate mapping of the Escherichia coli pepD gene by sequenceanalysis of its 5′ flanking region. 1989. Mol Gen Genet. 215:369-373);the pepE gene product from E. coli (Conlin C. A., and T. M. Knox, and C.G. Miller. Cloning and physical map position of an alpha-aspartyldepeptidase gene, pepE, from Escherichia coli. 1994. J Bacteriol.176:1552-1553); the pepN gene product from E. coli (Miller C. G., and G.Schwartz. Peptidase-deficient mutants of Escherichia coli. 1978. JBacteriiol. 135:603-611); the pepP gene product from E. coli (Id.); thepepQ gene product from E. coli (Id.); the pepT gene product from E. coli(Miller G. G., and G. Schwartz. Peptidase-deficient mutants ofEscherichia coli. 1978. J Bacteriiol. 135:603-611); the pilD geneproduct from E. coli (Francetic O., and S. Lory, and A. P. Pugsley. Asecond prepilin peptidase gene in Escherichia coli K-12. 1998, MolMicrobiol. 27:763-775); the pinA gene product from E. coli (Hilliard J.J., and L. D. Simon, and L. Van Melderen, and M. R. Maurizi. PinAinhibits ATP hydrolysis and energy-dependent protein degradation by Lonprotease. 1998. J Biol Chem. 273:524-527); the prc(tsp) gene productfrom E. coli (Nagasawa H., and Y. Sakagami, and A. Suzuki, and H.Suzuki, and H. Hara, and Y. Hirota. Determination of the cleavage siteinvolved in C-terminal processing of penicillin-binding proein 3 ofEscherichia coli. 1989. J Bacteriol. 171:5890-5893); the prlC geneproduct from E. coli (Jiang X., and M. Zhang, and Y. Ding, and J. Yao,and H. Chen, and D. Zhu, and M. Muramatu. Escherichia coli prlC geneencodes a trypsin-like proteinase regulating the cell cycle. 1998. JBiochem (Tokyo) 128:980-985); the protease V gene product from E. coli(Berlyn, M. K. B. et al. 1996. Linkage map of Escherichia coli K-12,Edition 9, In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin,K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, andH. E. Umbarger (eds.). Escherichia coli and Salmonella typhimurium:Cellular and Molecular Biology, 2^(nd) ed. American Society forMicrobiology, Washington, D.C.); the protease VI gene product from E.coli (Id.); the protease In gene product from E. coli (Id.); theprotease Fa gene product from E. coli or homologues (Id.); the proteaseMi gene product from E. coli (Id.); the protease So gene product from E.coli (Id.); the ptrA gene product from E. coli (Id.); the ptrB geneproduct from E. coli (Id.); the sypB gene product from E. coli (BarendsS., and A. W. Karzai, and R. T. Sauer, and J. Wower, and B. Kraal.Simultaneous an functional binding of SmpB and EF-Tu-TP to the analylacceptor arm of tmRNA. 2001. J Mol Biol. 314:9-21); the sohB geneproduct from E. coli (Baird L., and B. Lipinska, and S. Raina, and C.Georgopoulos. Identification of the Escherichia coli sohB gene, amulticopy suppressor of the HtrA (DegP) null phenotype. 1991. JBacteriol. 173-5763-5770); the sspA gene product from E. coli (IchiharaS., and T. Suzuki, and M. Suzuki, and C. Mizushima. Molecular cloningand sequencing of the sppA gene and characterization of the encodedproteas IV, a signal peptide peptidase of Escherichia coli. 1986. J BiolChem. 261;9405-9411); the tesA gene product from E. coli (Cho H., and J.E. Cronan Jr. Escherichia coli thioesterase I, molecular cloning andsequencing of the structural gene and identification as a periplasmicenzyme. 1993 J Biol Chem. 268:9238-9245); the tufA gene product from E.coli (Ang., and J. S. Lee, and J. D. Friesen. Evidence for an internalpromoter preceding tufA in the str operon of Escherichia coli. JBacteriol. 149:548-553); the tufB gene product from E. coli (MihajimaA., and M. Shibuya, and Y. Kaziro. Construction and characterization ofthe two hybrid Col1E1 plasmids carrying Escherichia coli tufB gene.1979. FEBS Lett. 102:207-210); the ybaU gene product from E. coli(Berlyn, M. K. B., et al. 1996. Linkage map of Escherichia coli K-12,Edition 9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin,K. B. Low, B. Magasanik, W. S. Resnikoff, M. Riley, M. Schaechter, andH. E. Umbarger (eds.). Escherichia coli and Salmonella typhimurium:Cellular and Molecular Biology, 2^(nd) ed. American Society forMicrobiology, Washington, D.C.); the ssrA gene (tmRNA, 10sA RNA) productfrom E. coli (Oh B. K., and A. K. Chauhan, and K. Isono, and D. Apirion.Location of a gene (ssrA) for a small, stable RNA 910Sa RNA) in theEscherichia coli chromosome. 1990. J Bacteriol. 172:4708-4709); and thessrB gene from E. coli (Berlyn, M. K. B., et al. 1996. Linkage map ofEscherichia coli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L.Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M.Riley, M. Schaechter, and H. E. Ummbarger 9eds.). Escherichia coli andSalmonella typhimurium: Cellular and Molecular Biology, 2^(nd) ed.American Society for Microbiology, Washington, D.C.).

[0396] II.C.13. Chaperones

[0397] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and functional proteinproduction in minicells. By way of non-limiting example, thesetechniques may include modification of chaperones and chaperonins, i.e.,endogenous and/or exogenous protein components that monitor the unfoldedstate of translated proteins allowing proper folding and/or secretion,membrane insertion, or soluble multimeric assembly of expressed proteinsin the parental cell prior to minicell formation and/or the segregatedminicell cytoplasm, membrane, periplasm, and/or extracellularenvironment. See Gottesman et al., Protein folding and unfolding byEscherichia coli chaperones and chaperonins, Current Op. Microbiol.3:197-202, 2000; and Mayhew et al., “Molecular Chaperone Proteins,”Chapter 61 in: Escherichia coli and Salmonella typhimurium: Cellular andMolecular Biology, 2nd Ed., Neidhardt, Frederick C., Editor in Chief,American Society for Microbiology, Washington, D.C., 1996, Volume 1,pages 922-937, and references cited therein.

[0398] These applications may, but are not limited to increased ordecreased chaperone production, increased or decreased intramolecularactivity of a chaperone, increased or decreased physiological functionof a chaperone, or deletion, substitution, inversion, translocation orinsertion into, or mutation of, a gene encoding a chaperone. By way ofnon-limiting example, increased production of a chaperone may occurthrough increased chaperone gene dosage (increased copy number of agiven gene under the control of the native or artificial promotor wherethis gene may be on a plasmid or in more than one copy on thechromosome), modification of the native regulatory elements, including,but not limited to the promotor or operator region(s) of DNA, orribosomal binding sites on RNA, relevant repressors/silencers, relevantactivators/enhancers, or relevant antisense nucleic acid or nucleic acidanalog, cloning on a plasmid under the control of the native orartificial promotor, and increased or decreased production of native orartificial promotor regulatory element(s) controlling production of thechaperone gene or gene product. By way of non-limiting example,decreased production of a chaperone may occur through modification ofthe native regulatory elements, including, but not limited to thepromotor or operator region(s) of DNA, or ribosomal binding sites onRNA, relevant repressors/silencers, relevant activators/enhancers, orrelevant antisense nucleic acid or nucleic acid analog, through cloningon a plasmid under the control of the native regulatory regioncontaining mutations or an artificial promotor, either or both of whichresulting in decreased chaperone production, and through increased ordecreased production of native or artificial promotor regulatoryelement(s) controlling production of the chaperone gene or gene product.By definition, intramolecular activity refers to the enzymatic function,structure-dependent function, e.g. the capacity of chaperone to interactin a protein-protein, protein-nucleic acid, or protein-lipid complex,and/or carrier function, e.g. the capacity to bind, either covalently ornon-covalently small organic or inorganic molecules, protein(s),carbohydrate(s), fatty acid(s), lipid(s), and nucleic acid(s). By way ofnon-limiting example, alteration of intramolecular activity may beaccomplished by mutation of the chaperone gene, in vivo or in vitrochemical modification of Chaperone, inhibitor molecules against thefunction of chaperone, e.g. competitive, non-competitive, oruncompetitive enzymatic inhibitors, inhibitors that preventprotein-protein, protein-nucleic acid, or protein-lipid interactions,e.g. expression or introduction of dominant-negative ordominant-positive chaperone or other protein fragment(s), or othercarbohydrate(s), fatty acid(s), lipid(s), and nucleic acid(s) that mayact directly or allosterically upon Chaperone, and/or modification ofprotein, carbohydrate, fatty acid, lipid, or nucleic acid moieties thatmodify the chaperone gene or gene product to create the functionalprotein. By definition, physiological function refers to the effectsresulting from an intramolecular interaction between Chaperone and otherprotein, carbohydrate, fatty acid, lipid, nucleic acid, or othermolecule(s) in or on the cell or the action of a product or productsresulting from such an interaction. By way of non-limiting example,physiological function may be the act or result of intermolecularphosphorylation, biotinylation, methylation, acylation, glycosylation,and/or other signaling event; this function may be the result of aprotein-protein, protein-nucleic acid, or protein-lipid interactionresulting in a functional moiety; this function may be to interact withthe membrane to recruit other molecules to this compartment of the cell;this function may be to regulate the transcription and/or translation ofchaperone, other protein, or nucleic acid; and this function may be tostimulate the function of another process that is not yet described orunderstood.

[0399] By way of non-limiting example, chaperone genes may be any of theE. coli genes listed below, as well as any homologs thereof fromprokaryotes, exukariutes, arcahebacteria, or organelles (mitochondria,chloroplasts, plastids, etc.). Exemplary E. coli genes encodingchaperones include, by way of non-limiting example, the cbpA gene(Shiozawa T., and C. Ueguchi, and T. Mizuno. The rpoD gene functions asa multicopy suppressor for mutations in the chaperones, CbpA, DnaJ andDnaK, in Escherichia coli. 1996 FEMS Microbiol Lett. 138:245-250): theclpB gene (Squires C. L., and S. Pedersen, and B. M. Ross, and C.Squires. ClpB is the Escherichia coli heat shock protein F84. 1. 1991. JBacteriol. 173:4254-4262); the dnaK gene (Kroczynska B., and S. Y.Blond. Cloning and characterization of a new soluble murine J-domainprotein that stimulates BiP, Hsc70 and DnaK ATPase activity withdifferent efficiencies. 2001. Gene. 273:267-274); the dnaJ gene(Kedzierska S., and E. Matuszewska. The effect of co-overproduction ofDnaK/DnaJ/GrpE and ClpB proteins on the removal of heat-aggregatedproteins from Escherichia coli Delta clpB mutant cells—new insight intothe role of Hsp70 in a functional cooperation with Hsp100. 2001. FEMSMicrobiol Lett. 204:355-360); the ecpD gene (Raina S., and D. Missiakas,and L. Baird, and S. Kumar, and C. Georgopoulos. Identification andtranscriptional analysis of the Escherichia coli htrE operon which ishomologous to pap and related pilin operons. 1993. J Bacteriol.175:5009-5021); the ffh gene (Muller, M., et al. 1002. Protein trafficin bacteria: multiple routes from the ribosome to and across themembrane. Prog. Nucleic Acid Res. Mol. Biol. 66:107-157); 4.5S RNA(Muller, M., et al. 1002. Protein traffic in bacteria: multiple routesfrom the ribosome to and across the membrane. Prog. Nucleic Acid Res.Mol. Biol. 66:107-157); the FtsY gene (Muller, M., et al. 1002. Proteintraffic in bacteria: multiple routes from the ribosome to and across themembrane. Prog. Nucleic Acid Res. Mol. Biol. 66:107-157); the fimC gene(Klemm P., and B. J. Jorgensen, and I. van Die, and H. de Ree, and H.Bergmans. The fim genes responsible for synthesis of type 1 fimbriae inEscherichia coli, cloning and genetic organization. 1985. Mol Gen Genet.199:410-414); the groe gene (Burton Z. F., and D. Eisenberg. A procedurefor rapid isolation of both groE protein and glutamine synthetase from Ecoli. 1980. Arch Biochem Biophys. 205:478-488); the groL gene (Berlyn,M. K. B., et al. 1996. Linkage map of Escherichia coli K-12, Edition 9.In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low,B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E.Umbarger (eds.). Escherichia coli and Salmonella typhimurium: cellularand molecular biology, 2nd ed. American Society for Microbiology,Washington D.C.); the groS gene (Berlyn, M. K. B., et al. 1996. Linkagemap of Escherichia coli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss,J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff,M. Riley, M. Schaechter, and H. E. Umbarger (eds.). Escherichia coli andSalmonella typhimurium: cellular and molecular biology, 2nd ed. AmericanSociety for Microbiology, Washington D.C.); the hptg gene (Berlyn, M. K.B., et al. 1996. Linkage map of Escherichia coli K-12, Edition 9. In F.C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B.Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger(eds.). Escherichia coli and Salmonella typhimurium: cellular andmolecular biology, 2nd ed. American Society for Microbiology, WashingtonD.C.); the hscA gene (Takahashi Y., and M. Nakamura. Functionalassignment of the ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene clusterinvolved in the assembly of Fe-S clusters in Escherichia coli. 1999. JBiochem (Tokyo). 126:917-926); the ibpA gene (Lund P. A. Microbialmolecular chaperones. 2001. Adv Microb Physiol. 44:93-140); the papJgene (Tennent, J. M., et al. 1990. Integrity of Escherichia coli P piliduring biogenesis: properties and role of PapJ. Mol. Microbiol.4:747-758); the secB gene (Lecker, S., et al. 1989. Three pure chaperoneproteins of Escherichia coli—SecB, trigger factor and GroEL—form solublecomplexes with precursor proteins in vitro. EMBO J. 8:2703-2709); andthe tig gene (Lecker, S., et 1989. Three pure chaperone proteins ofEscherichia coli—SecB, trigger factor and GroEL—form soluble complexeswith precursor proteins in vitro. EMBO J. 8:2703-2709); the secE gene(Muller, M., et al. 1002. Protein traffic in bacteria: multiple routesfrom the ribosome to and across the membrane. Prog. Nucleic Acid Res.Mol. Biol. 66:107-157); and the secY gene (Muller, M., et al. 1002.Protein traffic in bacteria: multiple routes from the ribosome to andacross the membrane. Prog. Nucleic Acid Res. Mol. Biol. 66:107-157).

[0400] II. C. 14. Export Apparatus and Membrane Targeting

[0401] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inparental cells prior to minicell formation and/or in the segregatedminicells. By way of non-limiting example, these techniques may includeconstruction of chimeric proteins including, but not limited to,coupling the expressed protein of interest with native Eubacterial,Eukaryotic, Archeabacterial or organellar leader sequences to drivemembrane insertion or secretion of the protein of interest to theperiplasm or extracellular environment. In addition to using nativeleader sequences, these minicell expression constructs may also includeproteolytic cleavage sites to remove the leader sequence followinginsertion into the membrane or secretion. These proteolytic cleavagesites may be native to the organism from which the minicell is derivedor non-native. In the latter example, also included in the system arethe non-native protease that recognizes the non-native proteolyticcleavage site.

[0402] Non-limiting examples of these leader sequences may be the leaderfrom the STII protein (Voss, T., et al. 1994. Periplasmic expression ofhuman interferon-alpha 2c in Escherichia coli results in a correctlyfolded molecule. Biochem. J. 298:719-725), maltose binding protein(malE) (Ito, K. 1982. Purification of the precursor form ofmaltose-binding protein, a periplasmic protein of Escherichia coli. J.Biol. Chem. 257:9895-9897), phoA (Jobling, M. G., et al. 1997.Construction and characterization of versatile cloning vectors forefficient delivery of native foreign proteins to the periplasm ofEscherichia coli. Plasmid. 38:158-173), lamB (Wong, E. Y., et al. 1988.Expression of secreted insulin-like growth factor-1 in Escherichia coli.Gene. 68:193-203), ompA (Loo, T., et al. 2002. Using secretion to solvea solubility problem: high-yield expression in Escherichia coli andpurification of the bacterial glycoamidase PNGase F. Protein Expr.Purif. 24:90-98), or pelB (Molloy, P. E., et al. 1998. Production ofsoluble single-chain T-cell receptor fragments in Escherichia coli trxBmutants. Mol. Immunol. 35:73-81).

[0403] In addition to these leader sequences, mutations in the cellularexport machinery may be employed to increase the promiscuity of exportto display or export sequences with non-optimized leader sequences.Non-limiting examples of genes that may be altered to increase exportpromiscuity are mutations in secY (prlA4) (Derman, A. I., et al. 1993. Asignal sequence is not required for protein export in prlA mutants ofEscherichia coli. EMBO J. 12:879-888), and secE (Harris, C. R., and T.J. Silhavy. 1999. Mapping an interface of SecY (PrlA) and SecE (PrlG) byusing synthetic phenotypes and in vivo cross-linking. J. Bacteriol.181:3438-3444).

[0404] II. C. 15. Increasing Stability and Solubility

[0405] Included in the design of the invention are techniques thatincrease the efficiency of gene expression and protein production inparental cells prior to minicell formation and/or in the segregatedminicells. By way of non-limiting example, these techniques may includeconstruction of chimeric/fusion proteins including, but not limited to,coupling the expressed protein of interest with native Eubacterial,Eukaryotic, Archeabacterial or organellar solublizing sequences. As usedherein, “solublizing sequences” are complete or truncated amino acidsequences that increase the solubility of the expressed membrane proteinof interest. This increased solubility may be used to increase thelifetime of the soluble state until proper membrane insertion may takeplace. By way of non-limiting example, these soluble chimeric fusionproteins may be ubiquitin (Power, R. F., et al. 1990. High levelexpression of a truncated chicken progesterone receptor in Escherichiacoli. J. Biol. Chem. 265:1419-1424), thioredoxin (LaVallie, E. R., etal. 1993. A thioredoxin gene fusion expression system that circumventsinclusion body formation in the E. coli cytoplasm. Biotechnology (N. Y.)11:187-193; Kapust, R. B., and D. S. Waugh. 1999. Escherichia colimaltose-binding protein is uncommonly effective at promoting thesolubility of polypeptides to which it is fused. Protein Sci.8:1668-1674), the dsbA gene product (Winter, J., et al. 2001. Increasedproduction of human proinsulin in the periplasmic space of Escherichiacoli by fusion to DsbA. J. Biotechnol. 84:175-185), the SPG protein(Murphy, J. P., et al. 1992. Amplified expression and large-scalepurification of protein G′. Bioseparation 3:63-71), the malE geneproduct (maltose-binding protein) (Hampe, W., et al. 2000. Engineeringof a proteolytically stable human beta 2-adrenergicreceptor/maltose-binding protein fusion and production of the chimericprotein in Escherichia coli and baculovirus-infected insect cells. J.Biotechnol. 77:219-234; Kapust et al., Escherichia coli maltose-bindingprotein is uncommonly effective at promoting the solubility ofpolypeptides to which it is fused, Protein Sci. 8:1668-1674, 1999),glutathione-s-transferase (GST); and/or nuclease A (Meeker et al., Afusion protein between serum amyloid A and staphylococcalnuclease--synthesis, purification, and structural studies, Proteins30:381-387, 1998). In addition to these proteins, Staphylococcal proteinA, beta-galactosidase, S-peptide, myosin heavy chain, dihydrofolatereductase, T4 p55, growth hormone N terminus, E. coli Hemolysin A,bacteriophage lambda cII protein, TrpE, and TrpLE proteins may also beused as fusion proteins to increase protein expression and/or solubility(Makrides, Stratagies for Achieving High-Level Expression of Genes inEscherichia coli, Microbiol. Rev. 60:512-538).

III. Preparation of Minicells

[0406] III.A. Parent Cell Mutations

[0407] Although it has been reported that relatively few molecules ofendogenous RNA polymerase segregate into minicells (Shepherd et al.,Cytoplasmic RNA Polymerase in Escherichia coli, J Bacteriol 183:2527-34,2001), other reports and results indicate that many RNA Polymerasemolecules follow plasmids into minicells (Funnell and Gagnier, Partitionof P1 plasmids in Escherichia cole mukB chromosomal partition mutants, JBacteriol 177:2381-6, 1995). In any event, applicants have discoveredthat the introduction of an exogenous RNA polymerase tominicell-producing cells enhances expression of episomal elements inminicells. Such enhanced expression may allow for the successfulexpression of proteins in minicells, wherein such proteins are expressedpoorly or not at all in unmodified minicells. In order to maximize theamount of RNA transcription from episomal elements in minicells,minicell-producing cell lines that express an RNA polymerase specificfor certain episomal expression elements may be used. An example of anE. coli strain of this type, designated MC-T7, was created and used asis described in the Examples. Those skilled in the art will be able tomake and use equivalent strains based on the present disclosure andtheir knowledge of the art.

[0408] Minicell-producing cells may comprise mutations that augmentpreparative steps. For example, lipopolysaccharide (LPS) synthesis in E.coli includes the lipid A biosynthetic pathway. Four of the genes inthis pathway have now been identified and sequenced, and three of themare located in a complex operon that also contains genes involved in DNAand phospholipid synthesis. The rfa gene cluster, which contains many ofthe genes for LPS core synthesis, includes at least 17 genes. The rfbgene cluster encodes protein involved in O-antigen synthesis, and rfbgenes have been sequenced from a number of serotypes and exhibit thegenetic polymorphism anticipated on the basis of the chemical complexityof the O antigens. See Schnaitman and Klena, Genetics oflipopolysaccharide biosynthesis in enteric bacteria, Microbiol. Rev.57:655-82, 1993. When present, alone, or in combination, the rfb and omsmutations cause alterations in the eubacterial membrane that make itmore sensitive to lysozyme and other agents used to process minicells.Similarly, the rfa (Chen, L., and W. G. Coleman Jr. 1993. Cloning andcharacterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a generequired for lipopolysaccharide inner core synthesis. J. Bacteriol.175:2534-2540), lpcA (Brooke, J. S., and M. A. Valvano. 1996.Biosynthesis of inner core lipopolysaccharide in enteric bacteriaidentification and characterization of a conserved phosphoheptoseisomerase. J. Biol. Chem. 271:3608-3614), and lpcB (Kadrman, J. L., etal. 1998. Cloning and overexpression of glycosyltransferases thatgenerate the lipopolysaccharide core of Rhizobium leguminosarum. J.Biol. Chem. 273:26432-26440) mutations, when present alone or incombination, cause alterations in lipopolysaccharides in the outermembrane causing cells to be more sensitive to lysozyme and agents usedto process minicells. In addition, such mutations can be used to reducethe potential antigenicity and/or toxicity of minicells.

[0409] III.B. Culturing Conditions

[0410] Included in the design of the invention are the conditions togrow parental cells from which minicells will be produced. Non-limitingexamples herein are drawn to conditions for growing E. coli parentalcells to produce minicells derived from E. coli parental cells.Non-limiting examples for growth media may include rich media, e.g.Luria broth (LB), defined minimal media, e.g. M63 salts with definedcarbon, nitrogen, phosphate, magnesium, and sulfate levels, and complexminimal media, e.g. defined minimal media with casamino acid supplement.This growth may be performed in culture tubes, shake flasks (using astandard air incubator, or modified bioreactor shake flask attachment),or bioreactor. Growth of parental cells may include supplementedadditions to assist regulation of expression constructs listed in thesections above. These supplements may include, but are not limited todextrose, phosphate, inorganic salts, ribonucleic acids,deoxyribonucleic acids, buffering agents, thiamine, or other chemicalthat stimulates growth, stabilizes growth, serves as an osmo-protectant,regulates gene expression, and/or applies selective pressure tomutation, and/or marker selection. These mutations may include an aminoacid or nucleotide auxotrophy, while the selectable marker may includetransposable elements, plasmids, bacteriophage, and/or auxotrophic orantibiotic resistance marker. Growth conditions may also requiretemperature adjustments, carbon alternations, and/or oxygen-levelmodifications to stimulate temperature sensitive mutations found indesigned gene products for a given desired phenotype and optimizeculture conditions.

[0411] By way of non-limiting example, production of minicells andprotein production may occur by using either of two general approachesor any combination of each. First, minicells may be formed, purified,and then contained expression elements may be stimulated to producetheir encoded gene products. Second, parental cells, from which theminicells are derived, may be stimulated to express the protein ofinterest and segregate minicells simultaneously. Finally, any timingvariable of minicell formation and protein production may be used tooptimize protein and minicell production to best serve the desiredapplication. The two general approaches are shown in the sections below.

[0412] III.C. Manipulation of Genetic Expression in Minicell Production

[0413] Included in the design of the invention are methods that increasethe efficiency, rate and/or level of gene expression and proteinproduction in parent cells and/or minicells. Such methods include, butare not limited to, the following.

[0414] By way of non-limiting example, parental cells are grownovernight in the appropriate media. From this culture, the cells aresubcultured into the same media and monitored for growth. At theappropriate cell density, the cultures are induced for minicellproduction using any of the switching mechanisms discussed in sectionII.B. regulating any construct discussed in section II.A. Non-limitingexamples of this minicell-inducing switching mechanism may be the ileRgene product regulating the production of the hns minicell-inducing geneproduct or the meiR gene product regulating the production of the minbminicell-inducing gene product. Following minicell induction, theculture is allowed to continue growth until the desired concentration ofminicells is obtained. At this point, the mincells are separated fromthe parental cells as described in section II.E. Purified minicells areinduced for protein production by triggering the genetic switchingmechanism that segregated into the minicell upon separation from theparental cell. By way of non-limiting example, this genetic switchingmechanism may be any of those discussed in section I.B. regulating theproduction of any protein of interest. Furthermore, at this point orduring the production of minicells the peripheral gene expression,production, and assembly machinery discussed in section II.C. may betriggered to assist in this process. By way of non-limiting example, thegroEL complex may be triggered using the temperature sensitive lambda cIinducible system from a co-segregant plasmid to assist in the properassembly of the expressed protein of interest.

[0415] III.D. Separation of Minicells From Parent Cells

[0416] A variety of methods are used to separate minicells from parentcells (i.e., the cells from which the minicells are produced) in amixture of parent cells and minicells. In general, such methods arephysical, biochemical and genetic, and can be used in combination.

[0417] III.D. 1. Physical Separation of Minicells from Parent Cells

[0418] By way of non-limiting example, minicells are separated fromparent cells glass-fiber filtration (Christen et al., Gene 23:195-198,1983), and differential and zonal centrifugation (Barker et al., J. Gen.Microbiol. 111:387-396, 1979), size-exclusion chromatography, e.g.gel-filtration, differential sonication (Reeve, J. N., and N. H.Mendelson. 1973. Pronase digestion of amino acid binding components onthe surface of Bacillus subtilis cells and minicells. Biochem. Biophys.Res. Commun. 53:1325-1330), and UV-irradiation (Tankersley, W. G., andJ. M. Woodward. 1973. Induction and isoloation of non-replicativeminicells of Salmonella typhimuium and their use as immunogens in mice.Bacteriol. Proc. 97).

[0419] Some techniques involve different centrifugation techniques,e.g., differential and zonal centrifugation. By way of non-limitingexample, minicells may be purified by the double sucrose gradientpurification technique described by Frazer and Curtiss, Curr. TopicsMicrobiol. Immunol. 69:1-84, 1975. The first centrifugation involvesdifferential centrifugation, which separates parent cells from minicellsbased on differences in size and/or density. The percent of sucrose inthe gradient (graduated from about 5 to about 20%), Ficol or glycerol isdesigned to allow only parent cells to pass through the gradient.

[0420] The supernatant, which is enriched for minicells, is thenseparated from the pellet and is spun at a much higher rate (e.g.,≧11,000 g). This pellets the minicells and any parent cells that did notpellet out in the first spin. The pellet is then resuspended and layeredon a sucrose gradient.

[0421] The band containing minicells is collected, pelleted bycentrifugation, and loaded on another gradient. This procedure isrepeated until the minicell preparation is essentially depleted ofparent cells, or has a concentration of parent cells that is low enoughso as to not interfere with a chosen minicell application or activity.By way of non-limiting example, buffers and media used in these gradientand resuspension steps may be LB, defined minimal media, e.g. M63 saltswith defined carbon, nitrogen, phosphate, magnesium, and sulfate levels,complex minimal media, e.g. defined minimal media with casamino acidsupplement, and/or other buffer or media that serves as anosmo-protectant, stabilizing agent, and/or energy source, or may containagents that limit the growth of contaminating parental cells, e.g azide,antibiotic, or lack an auxotrophic supplemental requirement, e.g.thiamine.

[0422] Other physical methods may also be used to remove parent cellsfrom minicell preparations. By way of non-limiting example, mixtures ofparent cells and minicells are frozen to −20° C. and then thawed slowly(Frazer and Curtiss, Curr. Topics Microbiol. Immunol. 69:1-84, 1975).

[0423] III.D.2.Biochemical Separation of Minicells From Parent Cells

[0424] Contaminating parental cells may be eliminated from minicellpreparations by incubation in the presence of an agent, or under a setof conditions, that selectively kills dividing cells. Because minicellscan neither grow nor divide, they are resistant to such treatments.

[0425] Examples of biochemical conditions that prevent or kill dividingparental cells is treatment with a antibacterial agent, such aspenicillin or derivatives of penicillin. Penicillin has two potentialaffects. First, penicillin prevent cell wall formation and leads tolysis of dividing cells. Second, prior to lysis dividing cells formfilaments that may assist in the physical separation steps described insection III.E. 1. In addition to penicillin and its derivatives, otheragents may be used to prevent division of parental cells. Such agentsmay include azide. Azide is a reversible inhibitor of electrontransport, and thus prevents cell division. As another example,D-cycloserine or phage MS2 lysis protein may also serve as a biochemicalapproach to eliminate or inhibit dividing parental cells. (Markiewicz etal., FEMS Microbiol. Lett. 70:119-123, 1992). Khachatourians (U.S. Pat.No. 4,311,797) states that it may be desirable to incubateminicell/parent cell mixtures in brain heart infusion broth at 36° C. to38° C. prior to the addition of penicillin G and further incubations.

[0426] III.D.3. Genetic Separation of Minicells From Parent Cells

[0427] Alternatively or additionally, various techniques may be used toselectively kill, preferably lyse, parent cells. For example, althoughminicells can internally retain M13 phage in the plasmid stage of theM13 life cycle, they are refractory to infection and lysis by M13 phage(Staudenbauer et al., Mol. Gen. Genet. 138:203-212, 1975). In contrast,parent cells are infected and lysed by M13 and are thus are selectivelyremoved from a mixture comprising parent cells and minicells. A mixturecomprising parent cells and minicells is treated with M13 phage at anM.O.I.=5 (phage:cells). The infection is allowed to continue to a pointwhere ≧50% of the parent cells are lysed, preferably >75%, morepreferably ≧95% most preferably ≧99%; and ≧25% of the minicells arelysed or killed, preferably ≦15%, most preferably ≦1%.

[0428] As another non-limiting example of a method by which parent cellscan be selectively killed, and preferably lysed, a chromosome of aparent cell may include a conditionally lethal gene. The induction ofthe chromosomal lethal gene will result in the destruction of parentcells, but will not affect minicells as they lack the chromosomeharboring the conditionally lethal gene. As one example, a parent cellmay contain a chromosomal integrated bacteriophage comprising aconditionally lethal gene. One example of such a bacteriophage is anintegrated lambda phage that has a temperature sensitive repressor gene(e.g., lambda cI857). Induction of this phage, which results in thedestruction of the parent cells but not of the achromosomal minicells,is achieved by simply raising the temperature of the growth media. Apreferred bacteriophage to be used in this method is one that killsand/or lyses the parent cells but does not produce infective particles.One non-limiting example of this type of phage is one that lyses a cellbut which has been engineered to as to not produce capsid proteins thatare surround and protect phage DNA in infective particles. That is,capsid proteins are required for the production of infective particles.

[0429] As another non-limiting example of a method by which parent cellscan be selectively killed or lysed, toxic proteins may be expressed thatlead to parental cell lysis. By way of non-limiting example, theseinducible constructs may employ a system described in section II.B. tocontrol the expression of a phage holing gene. Holin genes fall with inat least 35 different families with no detectable orthologousrelationships (Grundling, A., et al. 2001. Holins kill without warning.Proc. Natl. Acad. Sci. 98:9348-9352) of which each and any may be usedto lyse parental cells to improve the purity of minicell preparations.

[0430] Gram negative eubacterial cells and minicells are bounded by aninner membrane, which is surrounded by a cell wall, wherein the cellwall is itself enclosed within an outer membrane. That is, proceedingfrom the external environment to the cytoplasm of a minicell, a moleculefirst encounters the outer membrane (OM), then the cell wall andfinally, the inner membrane (IM). In different aspects of the invention,it is preferred to disrupt or degrade the OM, cell wall or IM of aeubacterial minicell. Such treatments are used, by way of non-limitingexample, in order to increase or decrease the immunogenicity, and/or toalter the permeability characteristics, of a minicell.

[0431] Eubacterial cells and minicells with altered membranes and/orcell walls are called “poroplasts™” “spheroplasts,” and “protoplasts.”Herein, the terms “spheroplast” and “protoplast” refer to spheroplastsand protoplasts prepared from minicells. In contrast, “cellularspheroplasts” and “cellular protoplasts” refer to spheroplasts andprotoplasts prepared from cells. Also, as used herein, the term“minicell” encompasses not only minicells per se but also encompassesporoplasts™, spheroplasts and protoplasts.

[0432] In a poroplast, the eubacterial outer membrane (OM) and LPS havebeen removed. In a spheroplast, portions of a disrupted eubacterial OMand/or disrupted cell wall either may remain associated with the innermembrane of the minicell, but the membrane and cell wall is nonethelessporous because the permeability of the disrupted OM and cell wall hasbeen increased. A membrane is said to be “disrupted” when the membrane'sstructure has been treated with an agent, or incubated under conditions,that leads to the partial degradation of the membrane, therebyincreasing the permeability thereof. In contrast, a membrane that hasbeen “degraded” is essentially, for the applicable intents and purposes,removed. In preferred embodiments, irrespective of the condition of theOM and cell wall, the eubacterial inner membrane is not disrupted, andmembrane proteins displayed on the inner membrane are accessible tocompounds that are brought into contact with the minicell, poroplast,spheroplast, protoplast or cellular poroplast, as the case may be.

[0433] III.E.2. Poroplasts™

[0434] For various applications poroplasted minicells are capable ofpreserving the cytoplasmic integrity while producing increased stabilityover that of naked protoplasts. Maintenance of the cell wall inporoplasted minicells increases the osmotic resistance, mechanicalresistance and storage capacity over protoplasts while permittingpassage of small and medium size proteins and molecules through theporous cell wall. A poroplast is a Gram negative bacterium that has itsouter membrane only removed. The production of poroplasts involves amodification of the procedure to make protoplasts to remove the outermembrane (Birdsell et al., Production and ultrastructure of lysozyme andethylenediaminetetraacetate-Lysozyme Spheroplasts of Escherichia coli,J. Bacteriology 93: 427-437, 1967; Weiss, Protoplast formation inEscherichia coli. J. Bacteriol. 128:668-670, 1976). Like protoplasts,measuring the total LPS that remains in the poroplast preparation may beused to monitor the removal of the outer membrane. Endotoxin kits andantibodies reactive against LPS may be used to measure LPS in solution;increasing amounts of soluble LPS indicates decreased retention of LPSby protoplants. This assay thus makes it possible to quantify thepercent removal of total outer membrane from the poroplasted minicells.

[0435] Several chemical and physical techniques have been employed toremove the outer membrane of gram negative bacteria. Chemical techniquesinclude the use of EDTA in Tris to make cells susceptible to hydrophobicagents such as actinomycin C. Leive L. The barrier function of thegram-negative envelope. Ann N Y Acad Sci. May 1974 10;235(0):109-29.;Voll M J, Leive L. Actinomycin resistance and actinomycin excretion in amutant of Escherichia coli. J Bacteriol. May 1970 ;102(2):600-2; LacticAcid disruption of the outer membrane as measured by the uptake ofhydrophobic flourophores; Alakomi H L, Skytta E, Saarela M,Mattila-Sandholm T, Latva-Kala K, Helander I M. Lactic acidpermeabilizes gram-negative bacteria by disrupting the outer membrane.Appl Environ Microbiol. May 2000; 66(5):2001-5; and Polymyxin Bdisruption as measured by periplasmic constituent release (Teuber M,Cerny G. Release of the periplasmic ribonuclease I into the medium fromEscherichia coli treated with the membrane-active polypeptide antibioticpolymyxin B. FEBS Lett. May 1970 11;8(1):49-5 1). Physical techniquesinclude the use of osmodifferentiation to facilitate the disruption ofthe OM. Neu HC, Heppel L A. The release of enzymes from Escherichia coliby osmotic shock and during the formation of spheroplasts. J Biol Chem.September 1965; 240(9):3685-92. See also Voll M J, Leive L. Actinomycinresistance and actinomycin excretion in a mutant of Escherichia coli. JBacteriol. May 1970; 102(2):600-2; Fiil A, Branton D. Changes in theplasma membrane of Escherichia coli during magnesium starvation. JBacteriol. June 1969; 98(3):1320-7; and Matsuyama S, Fujita Y, MizushimaS. SecD is involved in the release of translocated secretory proteinsfrom the cytoplasmic membrane of Escherichia coli. EMBO J. January 1993;12(1):265-70.

[0436] III. E.3. Spheroplasts

[0437] A spheroplast is a bacterial minicell that has a disrupted cellwall and/or a disrupted OM. Unlike eubacterial minicells and poroplasts,which have a cell well and can thus retain their shape despite changesin osmotic conditions, the absence of an intact cell wall inspheroplasts means that these minicells do not have a rigid form.

[0438] III.E.4. Protoplasts

[0439] A protoplast is a bacterium that has its outer membrane and cellwall removed. The production of protoplasts involves the use of lysozymeand high salt buffers to remove the outer membrane and cell wall(Birdsell et al., Production and ultrastructure of lysozyme andethylenediaminetetraacetate-Lysozyme Spheroplasts of Escherichia coli,J. Bacteriology 93: 427-437, 1967; Weiss, Protoplast formation inEscherichia coli. J. Bacteriol. 128:668-670, 1976). Various commerciallyavailable lysozymes can be used in such protocols. Measuring the totalLPS that remains in the protoplast preparation is used to monitor theremoval of the outer membrane. Endotoxin kits assays can be used tomeasure LPS in solution; increasing amounts of soluble LPS indicatesdecreased retention of LPS by protoplasts. This assay thus makes itpossible to quantify the percent removal of total outer membrane fromthe minicells. Endotoxin assays are commerically available from, e.g.,BioWhittaker Molecular Applications (Rockland, Me.)

[0440] For minicell applications that utilize bacterial-derivedminicells, it may be necessary to remove the outer membrane ofGram-negative cells and/or the cell wall of any bacterial-derivedminicell. For Gram-positive bacterial cells, removal of the cell wallmay be easily accomplished using lysozyme. This enzyme degrades the cellwall allowing easy removal of now soluble cell wall components from thepelletable protoplasted minicells. In a more complex system, the cellwall and outer membrane of Gram-negative cells may be removed bycombination treatment with EDTA and lysozyme using a step-wise approachin the presence of an osmoprotecting agent (Birdsell, et al. 1967.Production and ultrastructure of lysozyme andethylenediaminetetraacetate-lysozyme spheroplasts of E. coli, J.Bacteriol. 93:427-437; Weiss, 1976. Protoplast formation in E. coli. J.Bacteriol. 128:668-670). By non-limitingBy way of non-limiting example,this osmoprotectant may be sucrose and/or glycerol. It has been foundthat the concentration of the osmoprotectant sucrose, the cell walldigesting enzyme lysozyme, and chelator EDTA can be optimized toincrease the quality of the protoplasts produced. Separation of eitherprepared Gram-negative spheroplasts prepared in either fashion fromremoved remaining LPS may occur through exposure of the spheroplastmixture to an anti-LPS antibody. By non-limitingBy way of non-limitingexample, the anti-LPS antibody may be covalently or non-covalentlyattached to magnetic, agarose, sepharose, sepheracyl, polyacrylamide,and/or sephadex beads. Following incubation, LPS is removed from themixture using a magnet or slow centrifugation resulting in aprotoplast-enriched supernatant.

[0441] Monitoring loss of LPS may occur through dot-blot analysis ofprotoplast mixtures or various commercially available endotoxin kitassays can be used to measure LPS in solution; increasing amounts ofsoluble LPS indicates decreased retention of LPS by protoplasts. Thisimmuno assay may comprise a step of comparing the signal to a standardcurve in order to quantify the percent removal of total outer membranefrom the minicells. Other endotoxin assays, such as the LAL Systems fromBioWhittaker, are commercially available. LPS removal has been measuredby gas chromatography of fatty acid methyl esters. Alakomi H L, SkyttaE, Saarela M, Mattila-Sandholm T, Latva-Kala K, Helander I M. Lacticacid permeabilizes gram-negative bacteria by disrupting the outermembrane. Appl Environ Microbiol. May 2000;66(5):2001-5.

[0442] In order to reduce, preferably eliminate, in vivo antigenicpotential of minicells or minicell protoplasts, minicell protoplasts maybe treated to remove undesirable surface components. Minicellprotoplasts so treated are referred to as “denuded minicells” a termthat encompasses both spheroplasts and protoplasts. Denuding minicellsor minicell protoplasts is accomplished by treatment with one or moreenzymes or conditions that selectively or preferentially removes or makeless antigenic externally displayed proteins. As one non-limitingexample, the protease trypsin is used to digest exposed proteins on thesurface of these particles. In this example, the proteolytic activity oftrypsin may be modulated or terminated by the additional of a soybeantrypsin inhibitor. Non-limiting examples of other proteases thatadditionally or alternatively may be used include chymotrypsin, papain,elastase, proteinase K and pepsin. For some such proteases, it may benecessary to limit the extent of proteolysis by, e.g., using asuboptimal concentration of protease or by allowing the reaction toproceed for a suboptimal period of time. By the term “suboptimal,” it ismeant that complete digestion is not achieved under such conditions,even though the reactions could proceed to completion under other (i.e.,optimal) conditions.

[0443] It is sometimes preferred to use molecular genetic techniques tocreate mutant derivatives of exogenous proteins that (1) are resistantto the proteases or other enzymes used to prepare minicells and (2)retain the desired biological activity of the receptor that is desiredto be retained, i.e., the ability to bind one or more ligands ofinterest.

[0444] It is within the scope of the invention to have two or moreexogenous proteins expressed within and preferentially displayed byminicells in order to achieve combined, preferable synergistic,therapeutic compositions. Similarly, two or more therapeutic minicellcompositions are formulated into the same composition, or areadministered during the same therapeutic minicell compositions (i.e.,“cocktail” therapies). In other types of “cocktail” therapy, one or moretherapeutic minicell compositions are combined or co-administered withone or more other therapeutic agents that are not minicell compositionssuch as, e.g., organic compounds, therapeutic proteins, gene therapyconstructs, and the like.

[0445] III.F. Minicells from L-form Eubacteria

[0446] L-form bacterial strains may be used to prepare minicells and arepreferred in some embodiments of the invention. L-form bacterial strainsare mutant or variant strains, or eubacteria that have been subject tocertain conditions, that lack an outer membrane, a cell wall, aperiplasmic space and extracellular proteases. Thus, in L-formEubacteria, the cytoplasmic membrane is the only barrier between thecytoplasm and its surrounding environment. For reviews, see Grichko, V.P., et al. 1999. The Potential of L-Form Bacteria in Biotechnology, Can.J. Chem. Engineering 77:973-977; and Gumpert J., et al. 1998 Use of cellwall-less bacteria (L-forms) for efficient expression and secretion ofheterologous gene products. Curr Opin Biotechnol. 9:506-9.

[0447] Segregation of minicells from L-form eubacterial parent cellsallows for the generation of minicells that are at least partiallydeficient in barriers that lie outside of the cytoplasmic membrane, thusproviding direct access to components displayed on the minicellmembrane. Thus, depending on the strains and methods of preparationused, minicells prepared from L-form eubacterial parent cells will besimilar if not identical to various forms of poroplasts, spheroplastsand/or protoplasts. Displayed components that are accessible in L-formminicells include, but are not limited to, lipids, small molecules,proteins, sugars, nucleic acids and/or moieties that are covalently ornon-covalently associated with the cytoplasmic membrane or any componentthereof.

[0448] By way of non-limiting example, L-form Eubacteria that can beused in the methods of the invention include species of Escherichia,Streptomyces, Proteus, Bacillus, Clostridium, Pseudomonas, Yersinia,Salmonella, Enterococcus and Erwinia. See Onoda, T., et al. 1987.Morphology, growth and reversion in a stable L-form of Escherichia coliK12. J. Gen. Microbiol. 133:527-534; Inanova, E. H., et al. 1997. Effectof Escherichia coli L-form cytoplasmic membranes on the interactionbetween macrophages and Lewis lung carcinoma cells: scanning electronmicroscopy. FEMS Immunol. Med. Microbiol. 17:27-36; Onoda, T., et al.2000. Effects of calcium and calcium chelators on growth and morphologyof Escherichia coli L-form NC-7. J Bacteriol. 182:1419-1422; Innes, C.M., et al. 2001. Induction, growth and antibiotic production ofStreptomyces viridifaciens L-form bacteria. J Appl Microbiol.90:301-308; Ferguson, C. M., et al. 2000. An ELISA for the detection ofBacillus subtilis L-form bacteria confirms their symbiosis instrawberry. Lett Appl Microbiol. 31:390-394; Waterhouse R. N., et al.1994. An investigation of enumeration and DNA partitioning in Bacillussubtilis L-form bacteria. J Appl Bacteriol. 77:497-503; Hoischen, C., etal. 2002. Novel bacterial membrane surface display system using cellwall-less L-forms of Proteus mirabilis and Escherichia coli. Appl.Environ. Microbiol. 68:525-531; Rippmann, J. F., et al. 1998.Procaryotic expression of single-chain variable-fragment (scFv)antibodies: secretion in L-form cells of Proteus mirabilis leads toactive product and overcomes the limitations of periplasmic expressionin Escherichia coli. Appl. Environ. Microbiol. 64:4862-4869; Mahony, D.E., et al. 1988. Transformation of Clostridium perfringens L forms withshuttle plasmid DNA. Appl. Environ. Microbiol. 54:264-267); Kurona, M.,et al. 1983. Intergenus cell fusions between L-form cells of Pseudomonasaeruginosa and Escherichia coli. Biken. J.26:103-111; Ivanova, E., etal. 2000. Studies of the interactions of immunostimulated macrophagesand Yersinia enterocolitica O:8. Can. J. Microbiol. 46:218-228; Allan,E. J., et al. 1993. Growth and physiological characteristics of Bacillussubtilis L-forms. J. Appl. Bacteriol. 74:588-594; Allan, E. J. 1991.Induction and cultivation of a stable L-form of Bacillus subtilis. J.Appl. Bacteriol. 70:339-343; Nishikawa, F., et al. 1994. Protectivecapacity of L-form Salmonella typhimurium against murine typhoid inC3H/HeJ mice. Microbiol. Immunol. 38:129-137; Kita, E., et al. 1993.Isolation of a cytotoxin from L-form Salmonella typhimurium. FEMSMicrobiol. Lett. 109:179-184; Jass, J., et al. Growth and adhesion ofEnterococcus faecium L-forms. FEMS Microbiol. Lett. 115:157-162; andU.S. Pat. No. 6,376,245.

IV. Assaying Minicells

[0449] IV.A. Efficiency of Minicell Production

[0450] The level of minicell production will vary and may be evaluatedusing methods described herein. Relatively high levels of minicellproduction are generally preferred. Conditions in which about 40% ofcells are achromosomal have been reported (see, e.g., Hassan et al.,Suppression of initiation defects of chromosome replication in Bacillussubtilis dnaA and oriC-deleted mutants by integration of a plasmidreplicon into the chromosomes, J Bacteriol 179:2494-502, 1997).Procedures for identifying strains that give high yields of minicellsare known in the art; see, e.g., Clark-Curtiss and Curtiss III, Analysisof Recombinant DNA Using Escherichira coli Minicells, Meth. Enzol.101:347-362, 1983.

[0451] Minicell production can be assessed by microscopic examination oflate log-phase cultures. The ratio of minicells to normal cells and thefrequency of cells actively producing minicells are parameters thatincrease with increasing minicell production.

[0452] IV.B. Detecting Protein Synthesis in Minicells

[0453] Methods for detecting and assaying protein production are knownin the art. See, e.g., Clark-Curtiss and Curtiss III, Meth Enzol101:347-362, 1983. As an exemplary procedure, transformed E. coliminicell-producing cells are grown in LB broth with the appropriateantibiotic overnight. The following day the overnight cultures arediluted 1:50 in fresh media, and grown at 37° C. to mid-log phase. If itis desired to eliminate whole cells, an antibiotic that kills growing(whole) cells but not quiescent cells (minicells) may be used. Forexample, in the case of cells that are not ampicillin resistant,ampicillin (100 mg per ml is added), and incubation is allowed tocontinue for about 2 more hrs. Cultures are then centrifuged twice atlow speed to pellet most of the large cells. Minicells are pelleted byspinning 10 min at 10,000 rpm, and are then resuspended in M63 minimalmedia supplemented with 0.5% casamino acids, and 0.5 mM cAMP, or M9minimal medium supplemented with 1 mM MgSO₄, 0.1 mM CaCl₂, 0.05% NaCl,0.2% glucose, and 1 ng per ml thiamine. Labeled (³⁵S) methonine is addedto the minicells for about 15 to about 90 minutes, and minicells areimmediately collected afterwards by centrifugation for 10 min at 4° C.and 14,000 rpm. Cells are resuspended in 50 to 100 μg Laemmeli-buffer,and disrupted by boiling and vortexing (2 min for each step).Incorporation of ³⁵S-methionine was determined by measuring the amountof radioactivity contained in 1 μl of the lysate after precipitation ofproteins with trichloroacetic acid (TCA). Minicell lysates (50,000 to100,000 cpm per lane) are subjected to PAGE on, e.g., 10% polyacrylamidegels in which proteins of known size are also run as molecular weightstandards. Gels are fixed and images there of are generated by, e.g.,autoradiography or any other suitable detection systems.

[0454] IV.C. Evaluating the Therapeutic Potential of Minicells

[0455] Various methods are used at various stages of development of atherapeutic minicell composition to estimate their therapeuticpotential. As a non-limiting example, the therapeutic potential ofminicells displaying a receptor is examined as follows.

[0456] IV.C.1. Receptors

[0457] The specificity of, rate of association of, rate of dissociationof, and/or stability of complexes resulting from, receptor binding toits ligand can be measured in vitro using methods known in the art.

[0458] In the case of a sphingolipid binding receptor, such as an S1Preceptor, the ligand (S1P) is detectably labeled so that the specificityof, rate of formation of, and degree of stability of complexes resultingfrom the ligand-receptor binding can be examined by measuring the degreeand rate at which the labeled ligand is removed from solution due to itsbinding to minicells displaying the receptor. In order to avoidextraneous factors from influencing these experiments, they are carriedout in buffered solutions that are as free of contaminating substancesas possible. However, as is understood in the art, stabilizing agentssuch as BSA (bovine serum albumin) or protease inhibitors may bedesirably included in these experiments. In a preferred environment, asphingolipid binding receptor is the rat EDG-1, rat EDG-3, rat SCaMPERand human SCaMPER, the sequences of which are set forth herein.

[0459] Minicell compositions that bind sphingolipids with the desiredspecificity are identified from the preceding experiments. Typically,studies of ligand-receptor binding then proceed to studies in which thebinding capacity of promising minicell compositions is tested under invitro conditions that are increasingly more representative of in vivoconditions. For example, binding experiments are carried out in thepresence of sera or whole blood in order to determine the therapeuticpotential of minicell compositions in the presence of compounds that arepresent within the circulatory system of an animal.

[0460] IV.C.2. Molecular Sponge

[0461] Minicell compositions can also be tested for their ability tobind and/or interanlize toxic compounds. The therapeutic potential ofsuch capacity is evaluated using experiments in which detectably labeledderivatives of a toxic compound are present in the bloodstream of ananesthetized animal, which may a human. The blood of the animal isshunted out of the body and past a device that incorporates a minicellcomposition before being returned to the body. The device is constructedso that the blood contacts a semipermeable membrane that is in contactwith the minicell composition. By “semipermeable” it is meant thatcertain agents can be freely exchanged across the membrane, whereasothers are retained on one side of the membrane or the other. Forexample, the toxic compound of interest is able to cross thesemipermeable membrane, whereas minicells and blood cells are separatelyretained in their respective compartments. Detectably labeledderivatives of the toxic compound are present in the bloodstream of theanimal. The capacity of the minicells to take up the toxic compoundcorresponds with a reduction of the levels of detectably labeledmaterial in the blood and an increase in detectably labeled material inthe minicell composition.

[0462] The above types of minicell-comprising compositions, devices, andprocedures may be incorporated into ex vivo modalities such as ex vivogene therapy and dialysis machines. An “ex vivo modality” is one inwhich a biological sample, such as a blood sample, is temporarilyremoved from an animal, altered through in vitro manipulation, and thenreturned to the body. In “ex vivo gene therapy,” cells in the samplefrom the animal are transformed with DNA in vitro and then returned tothe body. A “dialysis machine” is a device in which a fluid such asblood of an animal is temporarily removed therefrom and processedthrough one or more physical, chemical, biochemical, binding or otherprocesses designed to remove undesirable substances including but arenot limited to toxins, venoms, overexpressed or overactive endogenousagents, and pathogens or molecules derived therefrom.

[0463] Intraminicellular co-expression of a second molecule that isdisplayed on the surface of minicells, and which is a ligand for abinding moiety that is immobilized, can optionally be used in order toremove minicells from the sample before it is returned to the body. Inthe latter aspect, minicells that bind undesirable substances arepreferably removed with the undesirable compound remaining bound to theminicells. Minicells that have been used for ex vivo gene therapy, butwhich have failed to deliver a nucleic acid to any cells in the sample,can be removed in a similar manner.

[0464] IV. C.3. Minicell-Solubilized Receptors

[0465] It is known in the art to use recombinant DNA technology toprepare soluble (hydrophilic) receptor fragments from receptors thatbind a bioactive ligand. Unlike the native, membrane-bound receptor,which is relatively insoluble in water (hydrophobic), soluble receptorfragments can be formulated for therapeutic delivery using techniquesthat are known to have been used to formulate soluble agents.

[0466] Typically, soluble receptor fragments are used to competitivelyinhibit the binding of the receptor to its ligand. That is, the solublereceptor fragments bind the ligand at the expense of the membrane-boundreceptor. Because less of the ligand is bound to its receptor, thecellular response to the ligand is attenuated. Common cellular responsesthat are desirably attenuated include but are not limited to the uptakeof an undesirable agent (e.g., a toxin, a pathogen, etc.) and activationof a signaling pathway having undesirable consequences (e.g.,inflammation, apoptosis, unregulated growth, etc.).

[0467] Preparing a soluble fragment derived from a receptor is nottrivial. Typically, the three dimensional structure of the receptor isnot known, and must be predicted based on homology with other receptorsor by using software that predicts the tertiary structure of apolypeptide based on its amino acid sequence. Using the hypotheticalstructure of the receptor, a series of polypeptides are prepared thatcomprise amino acid sequences from the receptor but lack regions thereofthat are thought to be membrane-anchoring or transmembrane domain(s) ofthe receptor. Some of the polypeptides prepared this way may be soluble,some may retain the binding activity of the receptor, and a few may haveboth characteristics. Members of the latter class of polypeptides aresoluble receptor fragments, some of which may be amenable to developmentas a therapeutic or diagnostic agent.

[0468] For any given receptor, there is always the possibility that noneof the soluble fragments derived from the receptor will specificallybind its ligand with sufficient affinity as to be thereapeuticallyeffective. Thus, in some instances, it may not be possible to prepare areceptor fragment that is both soluble and sufficiently biologicallyactive.

[0469] The minicells of the invention provide a “universal carrier” forreceptors that allows the hydrophobic receptors to be solubilized in thesense that, although they remain associated with a membrane, theminicell is a small, soluble particle. That is, as an alternative topreparing a set of polypeptides to see which, if any of them, are watersoluble receptor fragments, one may, using the teachings of thedisclosure, prepare soluble minicells that display the receptor.

[0470] IV.C.4.Reducing Toxicity

[0471] For in vivo use of minicells for the purposes of eliciting animmune response or for therapeutic and diagnostic applications involvingdelivery of minicells to a human or to an anima, it may be useful tominimize minicell toxicity by using endotoxin-deficient mutants ofparent cells. Without being limited to the following example,lipopolysaccharide (LPS) deficient E. coli strains could be conjugatedwith minicell producing cells to make parent cells lacking theendotoxin. LPS synthesis in E. coli includes the lipid A biosyntheticpathway. Four of the genes in this pathway have now been identified andsequenced, and three of them are located in a complex operon which alsocontains genes involved in DNA and phospholipid synthesis. The rfa genecluster, which contains many of the genes for LPS core synthesis,includes at least 17 genes. The rfb gene cluster encodes proteininvolved in O-antigen synthesis, and rfb genes have been sequenced froma number of serotypes and exhibit the genetic polymorphism anticipatedon the basis of the chemical complexity of the O antigens (Schnaittmanand Klena. 1993. Genetics of lipopolysaccharide biosynthesis in entericbacteria. Microbiol. Rev. 57:655-82). When present alone or incombination the rfb and oms mutations cause alterations in theeubacterial membrane that make it more sensitive to lysozyme and otheragents used to process minicells. Similarly, the rfa (Chen, L., and W.G. Coleman Jr. 1993. Cloning and characterization of the Escherichiacoli K-12 rfa-2 (rfaC) gene, a gene required for lipopolysaccharideinner core synthesis. J. Bacteriol. 175:2534-2540), lpcA (Brooke, J. S.,and M. A. Valvano. 1996. Biosynthesis of inner core lipopolysaccharidein enteric bacteria identification and characterization of a conservedphosphoheptose isomerase. J. Biol. Chem. 271:3608-3614), and lpcB(Kadrman, J. L., et al. 1998. Cloning and overexpression ofglycosyltransferases that generate the lipopolysaccharide core ofRhizobium leguminosarum. J. Biol. Chem. 273:26432-26440) mutations, whenpresent alone or in combination, cause alterations inlipopolysaccharides in the outer membrane causing cells to be moresensitive to lysozyme and agents used to process minicells. In addition,such mutations can be used to reduce the potential antigenicity and/ortoxicity of minicells.

[0472] Minicell-producing cells may comprise mutations that augmentpreparative steps. For example, lipopolysaccharide (LPS) synthesis in E.coli includes the lipid A biosynthetic pathway. Four of the genes inthis pathway have now been identified and sequenced, and three of themare located in a complex operon that also contains genes involved in DNAand phospholipid synthesis. The rfa gene cluster, which contains many ofthe genes for LPS core synthesis, includes at least 17 genes. The rfbgene cluster encodes protein involved in O-antigen synthesis, and rfbgenes have been sequenced from a number of serotypes and exhibit thegenetic polymorphism anticipated on the basis of the chemical complexityof the O antigens. See Schnaitman and Klena, Genetics oflipopolysaccharide biosynthesis in enteric bacteria, Microbiol. Rev.57:655-82, 1993. When present, alone, or in combination, the rfb and omsmutations cause alterations in the eubacterial membrane that make itmore sensitive to lysozyme and other agents used to process minicells.Similarly, the rfa (Chen, L., and W. G. Coleman Jr. 1993. Cloning andcharacterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a generequired for lipopolysaccharide inner core synthesis. J. Bacteriol.175:2534-2540), lpcA (Brooke, J. S., and M. A. Valvano. 1996.Biosynthesis of inner core lipopolysaccharide in enteric bacteriaidentification and characterization of a conserved phosphoheptoseisomerase. J. Biol. Chem. 271:3608-3614), and lpcB (Kadrman, J. L., etal. 1998. Cloning and overexpression of glycosyltransferases thatgenerate the lipopolysaccharide core of Rhizobium leguminosarum. J.Biol. Chem. 273:26432-26440) mutations, when present alone or incombination, cause alterations in lipopolysaccharides in the outermembrane causing cells to be more sensitive to lysozyme and agents usedto process minicells. In addition, such mutations can be used to reducethe potential antigenicity and/or toxicity of minicells.

V. Genetic Expression in Minicells

[0473] Various minicells of the invention use recombinant DNA expressionsystems to produce a non-eubacterial protein, which may be a membraneprotein that is preferably “displayed” on the surface of minicells, amembrane protein that projects portions not associtiated with a membranetowards the interior of a minicell, or a soluble protein present in theexterior of the minicells. By “displayed” it is meant that a protein ispresent on the surface of a cell (or minicell) and is thus in contactwith the external environment of the cell. Non-limiting examples ofdisplayed exogenous proteins of the invention include mammalianreceptors and fusion proteins comprising one or more transmembranedomains. In other aspects of the invention, minicells use expressionelements to produce bioactive nucleic acids from templates therefor.

[0474] V.A. Expression Systems

[0475] In vivo and in vitro protein expression systems provide a varietyof techniques that allow scientists to transcribe and translate aminoacid polypeptides proteins from recombinant DNA templates (Kaufman,Overview of vector design for mammalian gene expression. Mol Biotechnol,2001. 16: 151-160; and Kozak, Initiation of translation in prokaryotesand eukaryotes. Gene, 1999. 234: 187-208).

[0476] Although minicells are virtually depleted of chromosomal DNA(Tudor et al., Presence of nuclear bodies in some minicells ofEscherichia coli. J Bacteriol, 1969. 98: 298-299), it has been reportedthat minicells have all the elements required to express nucleotidesequences that are present in episomal expression elements therein(Levy, Very stable prokaryote messenger RNA in chromosomelessEscherichia coli minicells. Proc Natl Acad Sci USA, 1975. 72: 2900-2904;Hollenberg et al., Synthesis of high molecular weight polypeptides inEscherichia coli minicells directed by cloned Saccharomyces cerevisiae2-micron DNA. Gene, 1976. 1: 33-47; Crooks et al., Transcription ofplasmid DNA in Escherichia coli minicells. Plasmid, 1983. 10: 66-72;Clark-Curtiss, Analysis of recombinant DNA using Escherichia coliminicells. Methods Enzymol, 1983. 101: 347-362).

[0477] Preferred expression vectors and constructs according to theinvention are episomal genetic elements. By “episomal” it is meant thatthe expression construct is not always linked to a cell's chromosome butmay instead be retained or maintained in host cells as a distinctmolecule entity. Minicells can retain, maintain and express episomalexpression constructs such as, e.g., plasmids, bacteriophage, virusesand the like (Crooks et al., Plasmin 10:66-72, 1983; Clark-Curtiss,Methods Enzymology 101:347-62, 1983; Witkiewicz et al., Acta. Microbiol.Pol. A 7:21-24, 1975; Ponta et al., Nature 269:440-2, 1977). By“retained” it is meant that the episomal expression construct is atleast temporarily present and expressed in a host parent cell and/orminicell; by “maintained” it is meant that the episomal expressionconstruct is capable of autonomous- replication within a host parentcell and/or minicell. In the context of episomal elements, the term“contained” encompasses both “retained” and “maintained.” A preferredtype of an episomal element according to the invention is one that isalways an extrachromocomal element, or which is part of a chromosome butbecomes an extrachromosomal element before or during minicellproduction.

[0478] The fact that minicells do not contain chromosomal DNA but docontain episomal expression elements, such as plasmids, that can be usedas templates for RNA synthesis means that the only proteins that areactively produced in minicells are those that are encoded by theexpression elements that they contain. Minicell producing E. coli cellscan be made competent and transformed with expression elements thatdirect the expression of proteins encoded by the expression elements. Anexpression element segregates into minicells as they are produced. Inisolated minicells that contain expression elements, there is a singleDNA template RNA for transcription. Therefore, the only nucleic acidsand proteins that are actively produced (expressed) by minicells arethose that are encoded by sequences on the expression vector. In thecontext of the invention, sequences that encode amino acid sequences aredesignated “open reading frames” or “ORFs.” One feature of minicellexpression systems of interest as regards the present invention is thatendogenous (i.e., chromosomally located) genes are not present and arethus not expressed, whereas genes present on the episomal element areexpressed (preferably over-expressed) in the minicells. As a result, theamount of endogenous proteins, including membrane proteins, decreases asthe minicells continue to express genes located on episomal expressionconstructs.

[0479] The minicell system can reduce or eliminate undesirable featuresassociated with the transcription and translation of endogenous proteinsfrom the E. coli chromosome. For example, expression of proteins inminicell systems results in low background signal (“noise”) whenradiolabeled proteins produced using recombinant DNA technology(Jannatipour et al., Translocation of Vibrio HarveyiN,N′-DIacetylchitobiase to the outer membrane of Escherichia coli. J.Bacteriol, 1987. 169: 3785-3791). A high background signal can make itdifficult to detect a protein of interest. In whole cell E. colisystems, endogenous proteins (encoded by the bacterial chromosome) arelabeled as well as the protein(s) encoded by the expression element;whereas, in minicell systems, only the proteins encoded by theexpression element in the minicells are labeled.

[0480] There are a variety of proteins, both eubacterial and eukaryotic,that have been expressed from plasmid DNA in minicells (Clark-Curtiss,Methods Enzymal, 101:347-362, 1983). Some examples of proteins andnucleic acids that have been expressed in minicells include theKdp-ATPase of E. coli (Altendorf et al., Structure and function of theKdp-ATPase of Escherichia coli. Acta Physiol Scand, 643: 137-146, 1998);penicillin binding proteins aplha and gamma (Davies et al., Predictionof signal sequence-dependent protein translocation in bacteria:Assessment of the Escherichia coli minicell system. Biochem Biophys ResCommun, 150: 371-375, 1988); cell surface antigens of Polyronmaonasgingivalas (Rigg et al., The molecular cloning, nucleotide sequence andexpression of an antigenic determinant from Porphyromonas gingivalis.Arch Oral Biol, 45:41-52, 2000); trkG integral membrane protein of E.coli (Schlosser et al., Subcloning, Nucleotide sequence, and expressionof trkG, a gene that encodes an integral membrane protein involved inpotassium uptake via the Trk system of Escherichia coli. J. Bacteriol,173:3170-3176, 1991); the 34 kDa antigen of Treponema pallidum (Swancuttet al., Molecular characterization of the pathogen-specific,34-kilodalton membrane immunogen of Treponema pallidum. Infect Immun,57:3314-23, 1989); late proteins of bacteriophage MB78 (Colla et al.,IUBMB Life 48:493-497, 1999); uncharacterisized DNA from Xenopus laevis(Cohen and Boyer, U.S. Pat. No. 4,237,224, which issued Dec. 2, 1980);the onc gene v-fos (MacConnell and Verman, Expression of FBJ-MSVoncogene (fos) product in bacteria, 131(2) Virology 367 1983);interferon (Edge et al., Chemical synthesis of a human inteferon-alpha 2gene and its expression in Escherichia coli, Nucleic Acids Res. 11:6419,1983); bovine growth hormone (Rosner et al., Expression of a clonedbovine growth hormone gene in Escherichia coli minicells, Can. J.Biochem. 60:521-4, 1982); gastroitestinal hormone (Suzuki et al.,Production in Escherichia coli of biologically active secretin, agastroninstestinal hormone, Proc. Natl. Acad. Sci. USA 79:2475, 1982);and archeabacterial proteins (Lienard and Gottschalk, Cloning,sequencing and expression of the genes encoding the sodium translocatingN-methyltetrahydromethanopterin : coenzyme M methyltransferase of themethylotrohic archaeon Methanosarcina mazei Göl, 425 FEBS Letters 204,1998; and Lemker et al., Overproduction of a functional A1 ATPase fromthe archaeon Methanosarcina mazei G 1 in Escherichia coli, EuropeanJournal of Biochemistry 268:3744, 2001).

[0481] V.B. Modulating Genetic Expression in Minicells

[0482] Gene expression in minicells, and/or in minicell-producing(parent) cells, involves the coordinated activity of a variety ofexpression factors,regulatory elements and expression sequences. Any ofthese may be modified to alter the extent, timing or regulation ofexpression of a gene of interest in minicells and/or their parent cells.Often, the goal of the manipulations is to increase the efficiency ofprotein production in minicells. However, increased expression may, insome instances, desirably include increased or “tight” negativeregulation. This may reduce or eliminate selective pressure created bytoxic gene products, and allow for functional expression in a controlledfashion by removing the negative regulation and/or inducing expressionof the gene product at a preselected time. By way of non-limitingexample, these techniques may include modification or deletion ofendogenous gene(s) from which their respective gene product decreasesthe induction and expression efficiency of a desired protein in theparent cell prior to minicell formation and/or the segregated minicell.By way of non-limiting example, these protein components may be theenzymes that degrade chemical inducers of expression, proteins that havea dominant negative affect upon a positive regulatory elements, proteinsthat have proteolytic activity against the protein to be expressed,proteins that have a negative affect against a chaperone that isrequired for proper activity of the expressed protein, and/or thisprotein may have a positive effect upon a protein that either degradesor prevents the proper function of the expressed protein. These geneproducts that require deletion or modification for optimal proteinexpression and/or function may also be antisense nucleic acids that havea negative affect upon gene expression.

VI. Fusion (Chimeric) Proteins

[0483] In certain aspects of the invention, a fusion protein isexpressed and displayed by minicells. One class of fusion proteins ofparticular interest are those that are displayed on the surface ofminicells, e.g., fusion proteins comprising one or more transmembranedomains. Types of displayed fusion proteins of particular interest are,by way of non-limiting example, those that have an extracellular domainthat is a binding moiety or an enzymatic moiety. By way of non-limitingexample, the fusion protein ToxR-PhoA has been expressed in anddisplayed on the surface of minicells. The ToxR-PhoA fusion proteincomprises a polypeptide corresponding to the normally soluble enzyme,alkaline phosphatase, anchored to the minicell membrane by the singletransmembrane domain of ToxR (see the Examples). The fusion proteinretains the activity of the enzyme in the context of the minicellmembrane in which it is bound. Nearly all of the fusion protein isoriented so that the enzyme's catalytic domain is displayed on the outersurface of the minicell.

[0484] VI.A. Generation of Fusion Proteins

[0485] Polypeptides, which are polymers of amino acids, are encoded byanother class of molecules, known as nucleic acids, which are polymersof structural units known as nucleotides. In particular, proteins areencoded by nucleic acids known as DNA and RNA (deoxyribonucleic acid andribonucleic acid, respectively).

[0486] The nucleotide sequence of a nucleic acid contains the“blueprints” for a protein. Nucleic acids are polymers of nucleotides,four types of which are present in a given nucleic acid. The nucleotidesin DNA are adenine, cytosine and guanine and thymine, represented by A,C, G, and T respectively); in RNA, thymine (T) is replaced by uracil(U). The structures of nucleic acids are represented by the sequence ofits nucleotides arranged in a 5′ (“5 prime”) to 3′ (“3 prime”)direction, e.g.,

[0487] 5′-A-T-G-C-C-T-A-A-A-G-C-C-G-C-T-C-C-C-T-C-A-3′

[0488] In biological systems, proteins are typically produced in thefollowing manner. A DNA molecule that has a nucleotide sequence thatencodes the amino acid sequence of a protein is used as a template toguide the production of a messenger RNA (mRNA) that also encodes theprotein; this process is known as transcription. In a subsequent processcalled translation, the mRNA is “read” and directs the synthesis of aprotein having a particular amino acid sequence.

[0489] Each amino acid in a protein is encoded by a series of threecontiguous nucleotides, each of which is known as a codon. In the“genetic code,” some amino acids are encoded by several codons, eachcodon having a different sequence; whereas other amino acids are encodedby only one codon sequence. An entire protein (i.e., a complete aminoacid sequence) is encoded by a nucleic acid sequence called a readingframe. A reading frame is a continuous nucleotide sequence that encodesthe amino acid sequence of a protein; the boundaries of a reading frameare defined by its initiation (start) and termination (stop) codons.

[0490] The process by which a protein is produced from a nucleic acidcan be diagrammed as follows:

[0491] A chimeric reading frame encoding a fusion protein is prepared asfollows. A “chimeric reading frame” is a genetically engineered readingframe that results from the fusion of two or more normally distinctreading frames, or fragments thereof, each of which normally encodes aseparate polypeptide. Using recombinant DNA techniques, a first readingframe that encodes a first amino acid sequence is linked to a secondreading frame that encodes a second amino acid sequence in order togenerate a chimeric reading frame. Chimeric reading frames may alsoinclude nucleotide sequences that encode optional fusion proteinelements (see below).

[0492] A hypothetical example of a chimeric reading frame created fromtwo normally separate reading frames is depicted in the followingflowchart.

[0493] First Open Reading Frame and “Protein-1”

[0494] Second Open Reading Frame and “Protein-2”

[0495] Chimeric Reading Frame that Encodes a Fusion Protein havingSequences from Protein-1 and Protein-2

[0496] In order for a chimeric reading frame to be functional, eachnormally distinct reading frame therein must be fused to all of theother normally distinct reading frames in a manner such that all of thereading frames are in frame with each other. By “in frame with eachother” it is meant that, in a chimeric reading frame, a first nucleicacid having a first reading frame is covalently linked to a secondnucleic acid having a second reading frame in such a manner that the tworeading frames are “read” (translated) in register with each other. As aresult, the chimeric reading frame encodes one extended amino acidsequence that includes the amino acid sequences encoded by each of thenormally separate reading frames. A fusion protein is thus encoded by achimeric reading frame.

[0497] The fusion proteins of the invention are used to displaypolypeptides on minicells. The fusion proteins comprise (1) at least onepolypeptide that is desired to be displayed by minicells (a “displayedpolypeptide”) and (2) at least one membrane polypeptide, e.g., atransmembrane or a membrane anchoring domain. For various aspects of theinvention, optional fusion protein elements, as defined herein, may alsobe included if required or desired.

[0498] VI.B. Optional Fusion Protein Elements

[0499] The fusion proteins of the invention may optionally comprise oneor more non-biologically active amino acid sequences, i.e., optionalfusion protein elements. Such elements include, but are not limited to,the following optional fusion protein elements. It is understood that achimeric reading frame will include nucleotide sequences that encodesuch optional fusion protein elements, and that these nucleotidesequences will be positioned so as to be in frame with the reading frameencoding the fusion protein. Optional fusion protein elements may beinserted between the displayed polypeptide and the membrane polypeptide,upstream or downstream (amino proximal and carboxy proximal,respectively) of these and other elements, or within the displayedpolypeptide and the membrane polypeptide. A person skilled in the artwill be able to determine which optional element(s) should be includedin a fusion protein of the invention, and in what order, based on thedesired method of production or intended use of the fusion protein.

[0500] Detectable polypeptides are optional fusion protein elements thateither generate a detectable signal or are specifically recognized by adetectably labeled agent. An example of the former class of detectablepolypeptide is green fluorescent protein (GFP). Examples of the latterclass include epitopes such as a “His tag” (6 contiguous His residues,a.k.a. 6×His), the “FLAG tag” and the c-myc epitope. These and otherepitopes can be detected using labeled antibodies that are specific forthe epitope. Several such antibodies are commercially available.

[0501] Attachment (support-binding) elements are optionally included infusion proteins and can be used to attach minicells displaying a fusionprotein to a preselected surface or support. Examples of such elementsinclude a “His tag,” which binds to surfaces that have been coated withnickel; streptavidin or avidin, which bind to surfaces that have beencoated with biotin or “biotinylated” (see U.S. Pat. No. 4,839,293 andAirenne et al., Protein Expr. Purif. 17:139-145, 1999); andglutathione-s-transferase (GST), which binds to surfaces coated withglutathione (Kaplan et al., Protein Sci. 6:399-406, 1997; U.S. Pat. No.5,654,176). Polypeptides that bind to lead ions have also been described(U.S. Pat. No. 6,111,079).

[0502] Spacers (a.k.a. linkers) are amino acid sequences that areoptionally included in a fusion protein in between other portions of afusion protein (e.g., between the membrane polypeptide and the displayedpolypeptide, or between an optional fusion protein element and theremainder of the fusion protein). Spacers can be included for a varietyof reasons. For example, a spacer can provide some physical separationbetween two parts of a protein that might otherwise interfere with eachother via, e.g., steric hindrance. The ability to manipulate thedistance between the membrane polypeptide and the displayed polypeptideallows one to extend the displayed polypeptide to various distances fromthe surface of minicells.

[0503] VI.C. Interactions with Receipient Cells

[0504] Many Gram-negative pathogens use a type III secretion machine totranslocate protein toxins across the bacterial cell envelope (for areview, see Cheng L W, Schneewind O. Type III machines of Gram-negativebacteria: delivering the goods. Trends Microbiol May 2000;8(5):214-20).For example, pathogenic Yersinia spp. export over a dozen Yop proteinsvia a type III mechanism, which recognizes secretion substrates bysignals encoded in yop mRNA or chaperones bound to unfolded Yopproteins. A 70-kb virulence plasmid found in pathogenic Yersinia spp. tosurvive and multiply in the lymphoid tissues of the host. The virulenceplasmid encodes the Yop virulon, an integrated system allowingextracellular bacteria to inject bacterial proteins into cells. The Yopvirulon comprises a variety of Yop proteins and a dedicated type IIIsecretion apparatus, called Ysc (for a review, see Cornelis G R, BolandA, Boyd A P, Geuijen C, Iriarte M, Neyt C, Sory M P, Stainier I. Thevirulence plasmid of Yersinia, an antihost genome. Microbiol Mol BiolRev 1998 62(4):1315-52).

VII. Mincell Display

[0505] Included in the design of the invention is the use of minicellsto express and display soluble or membrane-bound protein libraries toidentify a soluble or membrane-bound protein that binds a known ligandor to identify proteins (e.g. orphan receptors) for which the knownligand or substrate is not known but for which a reporter could beengineered into the minicell that would signal the presence of theencoded protein. In the preferred embodiment of the invention, this‘minicell display’ technique is analogous to phage display for thepurpose of identifying genes that encode receptor-like or antibody-likeproteins against known ligand. This approach will allow identificationof an unknown receptor protein for which a known ligand has affinity.These known ligands may have been identified as having apharmacological, biological, or other effect without knowledge of thesite of effect. In these cases the knowledge of receptor will allowbasic research to understand the molecular and/or physiological responseand permit directed modification of the ligand for betterpharmacological or biological response or modification of the receptorfor employment in ligand-binding applications. In another non-limitingembodiment of the invention, the ligand need not be known but somegeneral characteristic of the protein would be.

[0506] For purposes of this application, soluble or membrane-boundprotein libraries may be constructed by random cloning of DNA fragmentsor directed cloning using reverse transcriptase polymerase chainreaction (RT-PCR). In either method, DNA fragments may be placed underthe regulation of any regulatory element listed in section II.B. on anyplasmid or chromosomal construct. In the case of soluble proteinreceptors, they will be fused to form a chimeric protein with a knowntransmembrane domain (TMD), e.g. the TMD from the toxR gene product.Upon induction of the soluble or membrane-bound protein library,minicells, minicell protoplasts, or minicell poroplasts (as theexperiment requires) will be mixed with the known ligand. Without beinglimited to the following example, screening could be accomplished byfirst labeling the known ligand with a molecular flourphore, e.g. TAMRA,FTC, or in some cases a fluorescent protein, e.g. GFP. A positiveinteraction between the minicells displaying the receptor for thelabeled ligand will be identified and separated from the librarypopulation by fluorescent-activated cell sorting (FACS). Isolated,positive receptor-ligand interactions will be identified by PCRamplification, subcloned into a clean background, and sequenced usingplasmid-specific oligonucleotides. Subcloned proteins will bere-screened for interaction with the labeled ligand, and their bindingpatterns characterized.

[0507] Positive interacting receptor proteins may be employed inmutagenesis or other directed evolutionary process to improve ordecrease the binding affinity to the ligand. In another application, thereceptor-ligand pair may be further employed in a screening process toidentify new compounds that may interfere with the interaction. Thus,using a known substance to identify the receptor and the identifiedreceptor-ligand pair to identify other interfering compounds.Chimeric-soluble or membrane-bound protein libraries may be screenedversus a protein-array chip that presents a variety of known proteincompounds or peptide variations. In this application, the minicell,minicell protoplast, or minicell poroplast will also contain a label,signaling component, and/or antigen recognizable by an antibody foridentification of a positive interaction on the protein chip array.Other approaches for identification may include packaged fluorescentmolecules or proteins that are constitutively produced, induced by thepositive interaction with the ligand, or regulated by a regulatoryelement described in section II.B.

[0508] In a preferred embodiment of the invention, cDNA libraries couldbe constructed from isolated B-cells, activated B-cell or T-cells forthe purpose of identifying receptors or antibodies that are encoded bythese cells of the immune system. In a non-limiting example, a smallmolecule could be used to immunize an experimental animal (e.g., rat,mouse, rabbit), the spleen could be removed, or blood could be drawn andused as a source of mRNA. Reverse transcription reactions could then beused to construct a cDNA library that would eventually be transformedinto the minicell parent bacteria, as described above. The minicellswould then be isolated, induced and subjected to FACS analysis withsubsequent amplification and sequencing of the cDNA fragment of interest(see above). The PCR-amplified plasmid-containing cDNA fragment encodingthe “receptor” or “antibody” of interest would be ready fortransformation and expression in the minicell context for diagnostic,therapeutic research or screening applications of the invention.

[0509] In a related, non-limiting embodiment of the invention, minicellsexpressing a particular antigen (e.g., protein, carbohydrate, smallmolecule, lipid) on their surfaces (described elsewhere in thisapplication) are used to generate an immunogenic response. Theadvantages of presenting an antigen on the surfaces of minicells arethat the minicells themselves may be an adjuvant that stimulates theimmune response, particularly if administered subcutaneously (SC) orintramuscularly (IM). Moreover, the minicells are not readily eliminatedby the renal system and are present in the circulatory system of animmunized animal for a longer time. In addition, small molecules couldbe tethered to the minicell in a way that presents the desired moiety ofthe molecule. Animals are presented with minicell-based immunogens, andthe antibodies produced in the animals are prepared and used intherapeutic, diagnostic, research and screening applications. Althoughthis aspect of the invention may be used to make antibodies to anymolecule displayed on their surface, the extracellular domains ofmembrane proteins are of particular interest.

[0510] Minicell display could be used to identify orphan receptors orother proteins for which a ligand or substrate is not known. As anon-limiting example, orphan G protein coupled receptors (GPCRs) ornovel RNA and DNA polymerases could be identified from organisms livingin extreme environments. A cDNA library could be is constructed from anorganism and expressed in minicells that co-express a reporter systemthat indicates the presence of the novel protein. In a non-limitingexample of GPCRs, the minicells used for minicell display are engineeredto express a G-protein in a manner that would signal an interaction withthe orphan GPCR.

VIII. Aptamers

[0511] Traditionally, techniques for detecting and purifying targetmolecules have used polypeptides, such as antibodies, that specificallybind such targets. While nucleic acids have long been known tospecifically bind other nucleic acids (e.g., ones having complementarysequences), aptamers (i.e., nucleic acids that bind non-nucleic targetmolecules) have been disclosed. See, e.g., Blackwell et al., Science(1990) 250:1104-1110; Blackwell et al., Science (1990) 250:1149-1152;Tuerk et al., Science (1990) 249:505-510; Joyce, Gene (1989) 82:83-87;and U.S. Pat. No. 5,840,867 entitled “Aptamer analogs specific forbiomolecules”.

[0512] As applied to aptamers, the term “binding” specifically excludesthe “Watson-Crick”-type binding interactions (i.e., A:T and G:Cbase-pairing) traditionally associated with the DNA double helix. Theterm “aptamer” thus refers to a nucleic acid or a nucleic acidderivative that specifically binds to a target molecule, wherein thetarget molecule is either (i) not a nucleic acid, or (ii) a nucleic acidor structural element thereof that is bound through mechanisms otherthan duplex- or triplex-type base pairing. Such a molecule is called a“non-nucleic molecule” herein.

[0513] VIII.A. Structures of Nucleic Acids

[0514] “Nucleic acids,” as used herein, refers to nucleic acids that areisolated a natural source; prepared in vitro, using techniques such asPCR amplification or chemical synthesis; prepared in vivo, e.g., viarecombinant DNA technology; or by any appropriate method. Nucleic acidsmay be of any shape (linear, circular, etc.) or topology(single-stranded, double-stranded, supercoiled, etc.). The term “nucleicacids” also includes without limitation nucleic acid derivatives such aspeptide nucleic acids (PNA's) and polypeptide-nucleic acid conjugates;nucleic acids having at least one chemically modified sugar residue,backbone, intemucleotide linkage, base, nucleoside, or nucleotideanalog; as well as nucleic acids having chemically modified 5′ or 3′ends; and nucleic acids having two or more of such modifications. Notall linkages in a nucleic acid need to be identical.

[0515] Nucleic acids that are aptamers are often, but need not be,prepared as oligonucleotides. Oligonucleotides include withoutlimitation RNA, DNA and mixed RNA-DNA molecules having sequences oflengths that have minimum lengths of 2, 4, 6, 8, 10, 11, 12, 13, 14, 15,17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides, and maximum lengths ofabout 100, 75, 50, 40, 25, 20 or 15 or more nucleotides, irrespectively.In general, a minimum of 6 nucleotides, preferably 10 nucleotides, morepreferably 14 to 20 nucleotides, is necessary to effect specificbinding.

[0516] In general, the oligonucleotides may be single-stranded (ss) ordouble-stranded (ds) DNA or RNA, or conjugates (e.g., RNA moleculeshaving 5′ and 3′ DNA “clamps”) or hybrids (e.g., RNA:DNA pairedmolecules), or derivatives (chemically modified forms thereof). However,single-stranded DNA is preferred, as DNA is often less labile than RNA.Similarly, chemical modifications that enhance an aptamer's specificityor stability are preferred.VIII.B. Chemical Modifications of NucleicAcids

[0517] Chemical modifications that may be incorporated into aptamers andother nucleic acids include, with neither limitation nor exclusivity,base modifications, sugar modifications, and backbone modifications.

[0518] Base modifications: The base residues in aptamers may be otherthan naturally occurring bases (e.g., A, G, C, T, U, 5MC, and the like).Derivatives of purines and pyrimidines are known in the art; anexemplary but not exhaustive list includes aziridinylcytosine,4-acetylcytosine, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine (5MC), N6-methyladenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acidmethylester, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid, and 2,6-diaminopurine. In addition to nucleicacids that incorporate one or more of such base derivatives, nucleicacids having nucleotide residues that are devoid of a purine or apyrimidine base may also be included in aptamers.

[0519] Sugar modifications: The sugar residues in aptamers may be otherthan conventional ribose and deoxyribose residues. By way ofnon-limiting example, substitution at the 2′-position of the furanoseresidue enhances nuclease stability. An exemplary, but not exhaustivelist, of modified sugar residues includes 2′ substituted sugars such as2′-O-methyl-, 2′-O-alkyl, 2′-O-allyl, 2′-S-alkyl, 2′-S-allyl,2′-fluoro-, 2′-halo, or 2′-azido-ribose, carbocyclic sugar analogs,alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside, ethylriboside or propylriboside.

[0520] Backbone modifications: Chemically modified backbones include, byway of non-limiting example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Chemicallymodified backbones that do not contain a phosphorus atom have backbonesthat are formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatom and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages, including without limitation morpholinolinkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones;formacetyl and thioformacetyl backbones; methylene formacetyl andthioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; and amide backbones.

[0521] VIII.C. Preparation and Identification of Aptamers

[0522] In general, techniques for identifying aptamers involveincubating a preselected non-nucleic target molecule with mixtures (2 to50 members), pools (50 to 5,000 members) or libraries (50 or moremembers) of different nucleic acids that are potential aptamers underconditions that allow complexes of target molecules and aptamers toform. By “different nucleic acids” it is meant that the nucleotidesequence of each potential aptamer may be different from that of anyother member, that is, the sequences of the potential aptamers arerandom with respect to each other. Randomness can be introduced in avariety of manners such as, e.g., mutagenesis, which can be carried outin vivo by exposing cells harboring a nucleic acid with mutagenicagents, in vitro by chemical treatment of a nucleic acid, or in vitro bybiochemical replication (e.g., PCR) that is deliberately allowed toproceed under conditions that reduce fidelity of replication process;randomized chemical synthesis, i.e., by synthesizing a plurality ofnucleic acids having a preselected sequence that, with regards to atleast one position in the sequence, is random. By “random at a positionin a preselected sequence” it is meant that a position in a sequencethat is normally synthesized as, e.g., as close to 100% A as possible(e.g., 5′-C-T-T-A-G-T-3′) is allowed to be randomly synthesized at thatposition (C-T-T-N-G-T, wherein N indicates a randomized position where,for example, the synthesizing reaction contains 25% each of A,T,C and G;or x% A, w% T, y% C and z%G, wherein x+w+y+z=100. In later stages of theprocess, the sequences are increasingly less randomized and consensussequences may appear; in any event, it is preferred to ultimately obtainan aptamer having a unique nucleotide sequence.

[0523] Aptamers and pools of aptamers are prepared, identified,characterized and/or purified by any appropriate technique, includingthose utilizing in vitro synthesis, recombinant DNA techniques, PCRamplification, and the like. After their formation, target:aptamercomplexes are then separated from the uncomplexed members of the nucleicacid mixture, and the nucleic acids that can be prepared from thecomplexes are candidate aptamers (at early stages of the technique, theaptamers generally being a population of a multiplicity of nucleotidesequences having varying degrees of specificity for the target). Theresulting aptamer (mixture or pool) is then substituted for the startingapatamer (library or pool) in repeated iterations of this series ofsteps. When a limited number (e.g., a pool or mixture, preferably amixture with less than 100 members, more preferably less than 10members, most preferably 1, of nucleic acids having satisfactoryspecificity is obtained, the aptamer is sequenced and characterized.Pure preparations of a given aptamer are generated by any appropriatetechnique (e.g., PCR amplification, in vitro chemical synthesis, and thelike).

[0524] For example, Tuerk and Gold (Science (1990) 249:505-510) disclosethe use of a procedure termed “systematic evolution of ligands byexponential enrichment” (SELEX). In this method, pools of nucleic acidmolecules that are randomized at specific positions are subjected toselection for binding to a nucleic acid-binding protein (see, e.g., PCTInternational Publication No. WO 91/19813 and U.S. Pat. No. 5,270,163).The oligonucleotides so obtained are sequenced and otherwisecharacterization. Kinzler, K. W., et al. (Nucleic Acids Res. (1989)17:3645-3653) used a similar technique to identify syntheticdouble-stranded DNA molecules that are specifically bound by DNA-bindingpolypeptides. Ellington, A. D., et al. (Nature (1990) 346: 818-822)disclose the production of a large number of random sequence RNAmolecules and the selection and identification of those that bindspecifically to specific dyes such as Cibacron blue.

[0525] Another technique for identifying nucleic acids that bindnon-nucleic target molecules is the oligonucleotide combinatorialtechnique disclosed by Ecker, D. J. et al. (Nuc. Acids Res. 21, 1853(1993)) known as “synthetic unrandomization of randomized fragments”(SURF), which is based on repetitive synthesis and screening ofincreasingly simplified sets of oligonucleotide analogue libraries,pools and mixtures (Tuerk et al., Science 249:505, 1990). The startinglibrary consists of oligonucleotide analogues of defined length with oneposition in each pool containing a known analogue and the remainingpositions containing equimolar mixtures of all other analogues. Witheach round of synthesis and selection, the identity of at least oneposition of the oligomer is determined until the sequences of optimizednucleic acid ligand aptamers are discovered.

[0526] Once a particular candidate aptamer has been identified through aSURF, SELEX or any other technique, its nucleotide sequence can bedetermined (as is known in the art), and its three-dimensional molecularstructure can be examined by nuclear magnetic resonance (NMR). Thesetechniques are explained in relation to the determination of thethree-dimensional structure of a nucleic acid ligand that binds thrombinin Padmanabhan et al., J. Biol. Chem. 24, 17651 (1993); Wang et al.,Biochemistry 32, 1899 (1993); and Macaya et al., Proc. Nat'l. Acad. Sci.USA 90, 3745 (1993). Selected aptamers may be resynthesized using one ormore modified bases, sugars or backbone linkages. Aptamers consistessentially of the minimum sequence of nucleic acid needed to conferbinding specificity, but may be extended on the 5′ end, the 3′ end, orboth, or may be otherwise derivatized or conjugated.

IX. Polypeptidic Binding Moieties

[0527] A variety of binding moities can be attached to a minicell of theinvention for a variety of purposes. In a preferred embodiment, thebinding moiety is directed to a ligand that is displayed by a cell intowhich it is desired to deliver the therapeutic content of a minicell.

[0528] IX.A. Antibodies and Antibody Derivatives

[0529] The term “antibody” is meant to encompass an immunoglobulinmolecule obtained by in vitro or in vivo generation of an immunogenicresponse, and includes polyclonal, monospecific and monoclonalantibodies, as well as antibody derivatives, e.g single-chain antibodyfragments (scFv). An “immunogenic response” is one that results in theproduction of antibodies directed to one or more proteins after theappropriate cells have been contacted with such proteins, or polypeptidederivatives thereof, in a manner such that one or more portions of theprotein function as epitopes. An epitope is a single antigenicdeterminant in a molecule. In proteins, particularly denatured proteins,an epitope is typically defined and represented by a contiguous aminoacid sequence. However, in the case of nondenatured proteins, epitopesalso include structures, such as active sites, that are formed by thethree-dimensional folding of a protein in a manner such that amino acidsfrom separate portions of the amino acid sequence of the protein arebrought into close physical contact with each other.

[0530] Wildtype antibodies have four polypeptide chains, two identicalheavy chains and two identical light chains. Both types of polypeptidechains have constant regions, which do not vary or vary minimally amongantibodies of the same class (i.e., IgA, IgM, etc.), and variableregions. Variable regions are unique to a particular antibody andcomprise an “antigen binding domain” that recognizes a specific epitope.Thus, an antibody's specificity is determined by the variable regionslocated in the amino terminal regions of the light and heavy chains.

[0531] As used herein, the term “antibody” encompasses derivatives ofantibodies such as antibody fragments that retain the ability tospecifically bind to antigens. Such antibody fragments include Fabfragments (i.e., an antibody fragment that contains the antigen-bindingdomain and comprises a light chain and part of a heavy chain bridged bya disulfide bond); Fab′ (an antibody fragment containing a singleanti-binding domain comprising an Fab and an additional portion of theheavy chain through the hinge region); F(ab′)2 (two Fab′ moleculesjoined by interchain disulfide bonds in the hinge regions of the heavychains; the Fab′ molecules may be directed toward the same or differentepitopes); a bispecific Fab (an Fab molecule having two antigen bindingdomains, each of which may be directed to a different epitope); a singlechain Fab chain comprising a variable region, a.k.a., a sFv (thevariable, antigen-binding determinative region of a single light andheavy chain of an antibody linked together by a chain of about 10 toabout 25 amino acids).

[0532] The term “antibody” includes antibodies and antibody derivativesthat are produced by recombinant DNA techniques and “humanized”antibodies. Humanized antibodies have been modified, by geneticmanipulation and/or in vitro treatment to be more human, in terms ofamino acid sequence, glycosylation pattern, etc., in order to reduce theantigenicity of the antibody or antibody fragment in an animal to whichthe antibody is intended to be administered (Gussow et al., Methods Enz.203:99-121, 1991).

[0533] A single-chain antibody (scFv) is a non-limiting example of abinding moiety that may be displayed on minicells. Single-chainantibodies are produced by recombinant DNA technology and may beincorporated into fusion proteins. The term “single chain” denotes thefact that scFv's are found in a single polypeptide. In contrast,wildtype antibodies have four polypeptide chains, two identical heavychains and two identical light chains. Both types of polypeptide chainshave constant regions, which do not vary or vary minimally amongantibodies of the same class (i.e., IgA, IgM, etc.), and variableregions. An antibody's specificity is determined by the variable regionslocated in the amino terminal regions of the light and heavy chains. Thevariable regions of a light chain and associated heavy chain form an“antigen binding domain” that recognizes a specific epitope. In a singlechain antibody, the amino acid sequences of the variable light andvariable heavy regions of an antibody are present in one contiguouspolypeptide. Methods of producing single chain antibodies are known inthe art. See, for example, U.S. Pat. Nos. 4,946,778; 5,260,203;5,455,030; 5,518,889; 5,534,621; 5,869,620; 6,025,165; 6,027,725 and6,121,424.

[0534] Antibody derivatives and other polypeptides that are bindingmoieties can be isolated from protein display libraries, in which alibrary of candidate binding agents is displayed on a phage or otheragent that comprises a nucleic acid encoding the protein it displays.Thus, an agent that binds to the target compound can be isolated, andnucleic acid prepared therefrom, providing for the rapid isolation ofbinding moieties and nucleic acids that can be used to produce them. Forreviews, see Benhar I. Biotechnological applications of phage and celldisplay. Biotechnology Adv. 2001 (19):1-33; FitzGerald K. In vitrodisplay technologies—new tools for drug discovery. Drug Discov Today.2000 5(6):253-258; and Hoogenboom H R, Chames P. Natural and designerbinding sites made by phage display technology. Immunol Today. August2000;21(8):371-8.

[0535] A variety of protein display systems are known in the art andinclude various phage display systems such as those described in Jung S,Arndt K, Müller K, Plückthyn A. Selectively infective phage (SIP)technology: scope and limitations. J Immunol Methods. 1999 (231):93-104;Katz B. Structural and mechanistic determinants of affinity andspecificity of ligands discovered or engineered by phage display. AnnuRev Biophys Biomol Struct. 1997 (26):27-45; Forrer P, Jung S, PluckthunA. Beyond binding: using phage display to select for structure, foldingand enzymatic activity in proteins. Curr Opin Struct Biol. August1999;9(4):514-20; Rondot S, Koch J, Breitling F, Dubel S. A helper phageto improve single-chain antibody presentation in phage display. NatBiotechnol. January 2001;19(1):75-8. Giebel L B, Cass R T, Milligan D L,Young D C, Arze R, Johnson C R. Screening of cyclic peptide phagelibraries identifies ligands that bind streptavidin with highaffinities. Biochemistry. Nov. 28, 1995;34(47):15430-5; de Kruif J,Logtenberg T. Leucine zipper dimerized bivalent and bispecific scFvantibodies from a semi-synthetic antibody phage display library. J BiolChem. Mar. 29, 1996;271(13):7630-4; Hoogenboom H R, Henderikx P, deHaard H. Creating and engineering human antibodies for immunotherapy.Adv Drug Deliv Rev. Apr. 6, 1998;31(1-2):5-31; Helfrich W, Haisma H J,Magdolen V, Luther T, Bom V J, Westra J, van der Hoeven R, Kroesen B J,Molema G, de Leij L. A rapid and versatile method for harnessing scfvantibody fragments with various biological effector functions. J ImmunolMethods. Apr. 3, 2000;237(1-2):131-45; Hoess R H. Bacteriophage lambdaas a vehicle for peptide and protein display. Curr Pharm BiotechnolMarch2002;3(1):23-8; Baek H, Suk K H, Kim Y H, Cha S. An improved helperphage system for efficient isolation of specific antibody molecules inphage display. Nucleic Acids Res. Mar. 1, 2002;30(5):e18; and Rondot S,Koch J, Breitling F, Dubel S. A helper phage to improve single-chainantibody presentation in phage display. Nat Biotechnol. January2001;19(1):75-8.

[0536] Other display systems include without limitation “Yeast Display”(Curr Opin Biotechnol October 1999;10(5):422-7. Applications of yeast inbiotechnology: protein production and genetic analysis. Cereghino G P,Cregg J M.); “Baculovirus Display” (Kost T A, Condreay J P. Recombinantbaculoviruses as expression vectors for insect and mammalian cells. CurrOpin Biotechnol. October 1999;10(5):428-33; and Liang M, Dubel S, Li D,Queitsch I, Li W, Bautz E K. Baculovirus expression cassette vectors forrapid production of complete human IgG from phage display selectedantibody fragments. J Immunol Methods. Jan. 1, 2001;247(1-2):119-30);“Ribosome Display” (Hanes J, Schaffitzel C, Knappik A, Pluckthun A.Picomolar affinity antibodies from a fully synthetic naive libraryselected and evolved by ribosome display. Nat Biotechnol. December2000;18(12): 1287-92; Hanes J, Jermutus L, Pluckthun A. Selecting andevolving functional proteins in vitro by ribosome display. MethodsEnzymol. 2000;328:404-30; Schaffitzel C, Hanes J, Jermutus L, PluckthunA. Ribosome display: an in vitro method for selection and evolution ofantibodies from libraries. J Immunol Methods. Dec. 10,1999;231(1-2):119-35; Hanes J, Jermutus L, Weber-Bornhauser S, BosshardH R, Pluckthun A. Ribosome display efficiently selects and evolveshigh-affinity antibodies in vitro from immune libraries. Proc Natl AcadSci U S A. Nov. 24, 1998;95(24):14130-5; Hanes J, Pluckthun A. In vitroselection and evolution of functional proteins by using ribosomedisplay. Proc Natl Acad Sci U S A. May 13, 1997;94(10):4937-42; Coia G,Pontes-Braz L, Nuttall S D, Hudson P J, Irving R A. Panning andselection of proteins using ribosome display. J Immunol Methods. Aug 1,2001;254(1-2):191-7.; Irving R A, Coia G, Roberts A, Nuttall S D, HudsonP J. Ribosome display and affinity maturation: from antibodies to singleV-domains and steps towards cancer therapeutics. J Immunol Methods. Feb.1, 2001;248(1-2):31-45); and “Bacterial Display” (Hoischen C, FritscheC, Gumpert J, Westermann M, Gura K, Fahnert B. Novel bacterial membranesurface display system using cell wall-less L-forms of Proteus mirabilisand Escherichia coli. Appl Environ Microbiol. February2002;68(2):525-31; Etz H, Minh D B, Schellack C, Nagy E, Meinke A.Bacterial phage receptors, versatile tools for display of polypeptideson the cell surface. J Bacteriol. December 2001;183(23):6924-35; PatelD, Vitovski S, Senior H J, Edge M D, Hockney R C, Dempsey M J, Sayers JR. Continuous affinity-based selection: rapid screening and simultaneousamplification of bacterial surface-display libraries. Biochem J. Aug. 1,2001;357(Pt 3):779-85; Lang H. Outer membrane proteins as surfacedisplay systems. Int J Med Microbiol. December 2000;290(7):579-85;Earhart CF. Use of an Lpp-OmpA fusion vehicle for bacterial surfacedisplay. Methods Enzymol. 2000;326:506-16; Benhar I, Azriel R, Nahary L,Shaky S, Berdichevsky Y, Tamarkin A, Wels W. Highly efficient selectionof phage antibodies mediated by display of antigen as Lpp-OmpA′ fusionson live bacteria. J Mol Biol. Aug 25, 2000;301(4):893-904; Xu Z, Lee SY. Display of polyhistidine peptides on the Escherichia coli cellsurface by using outer membrane protein C as an anchoring motif. ApplEnviron Microbiol. November 1999;65(11):5142-7; Daugherty P S, Olsen MJ, Iverson B L, Georgiou G. Development of an optimized expressionsystem for the screening of antibody libraries displayed on theEscherichia coli surface. Protein Eng. July 1999;12(7):613-21; Chang HJ, Sheu S Y, Lo S J. Expression of foreign antigens on the surface ofEscherichia coli by fusion to the outer membrane protein traT. J BiomedSci. January 1999;6(1):64-70; Maurer J, Jose J, Meyer T F. Autodisplay:one-component system for efficient surface display and release ofsoluble recombinant proteins from Escherichia coli. J Bacteriol.February 1997;179(3):794-804.

[0537] Antibodies, particularly single-chain antibodies, directed tosurface antigens specific for a particular cell type may also be used ascell- or tissue-specific targeting elements. Single-chain antibody aminoacid sequences have been incorporated into a variety of fusion proteins,including those with transmembrane domains and/or membrane-anchoringdomains. See, for example, Kuroki et al., “Specific Targeting Strategiesof Cancer Gene Therapy Using a Single-Chain Variable Fragment (scFv)with a High Affinity for CEA,” Anticancer Res., pp. 4067-71, 2000; U.S.Pat. No. 6,146,885, to Dornburg, entitled “Cell-Type Specific GeneTransfer Using Retroviral Vectors Containing Antibody-Envelope FusionProteins”; Jiang et al., “In Vivo Cell Type-Specific Gene Delivery WithRetroviral Vectors That Display Single Chain Antibodies,” Gene Ther.1999, 6:1982-7; Engelstadter et al., “Targeting Human T Cells ByRetroviral Vectors Displaying Antibody Domains Selected From A PhageDisplay Library,” Hum. Gene Ther. 2000, 11:293-303; Jiang et al.,“Cell-Type-Specific Gene Transfer Into Human Cells With RetroviralVectors That Display Single-Chain Antibodies,” J. Virol1998,72:10148-56; Chu et al., “Toward Highly EfficientCell-Type-Specific Gene Transfer With Retroviral Vectors DisplayingSingle-Chain Antibodies,” J. Virol 1997, 71:720-5; Chu et al.,“Retroviral Vector Particles Displaying The Antigen-Binding Site Of AnAntibody Enable Cell-Type-Specific Gene Transfer,” J. Virol 1995,69:2659-63; Chu et al., “Cell Targeting With Retroviral Vector ParticlesContaining Antibody-Envelope Fusion Proteins,” Gene Ther. 1994, 1:292-9;Esshar et al., “Specific activation and targeting of cytotoxiclymphocytes through chimeric single chains consisting ofantibody-binding domains and the or subunits of the immunoglobulin andT-cell receptors,” Proc. Natl. Acad. Sci. USA, 1993, Vol. 90:720-724;Einfeld et al., “Construction of a Pseudoreceptor That MediatesTransduction by Adenoviruses Expressing a Ligand in Fiber or PentonBase,” J. Virol. 1999, 73:9130-9136; Marin et al., “Targeted Infectionof Human Cells via Major Histocompatibility Complex Class I Molecules byMoloney Murine Leukemia Virus-Derived Viruses Displaying Single-ChainAntibody Fragment-Envelope Fusion Proteins,” J. Virol., 1996,70:2957-2962; Somia et al., “Generation of targeted retroviral vectorsby using single-chain variable fragment: An approach to in vivo genedelivery,” Proc. Natl. Acad. Sci. USA, 1995, 92:7570-7574; Liu et al.,“Treatment of B-Cell Lymphoma With Chimeric IgG and Single-Chain FvAntibody-Interleukin-2 Fusion Proteins,” Blood, 1998, 92:2103-2112;Martin et al., “Retrovirus Targeting by Tropism Restriction to MelanomaCells,” J. Virol., 1999, 73:6923-6929; Ramjiawan et al., “NoninvasiveLocalization of Tumors by Immunofluorescence Imaging Using a SingleChain Fv Fragment of a Human Monoclonal Antibody with Broad CancerSpecificity,” Amer. Cancer Society, 2000, 89:1134-30 1144; Snitkovsky etal., “A TVA-Single-Chain Antibody Fusion Protein Mediates SpecificTargeting of a Subgroup A Avian Leukosis Virus Vector to CellsExpressing a Tumor-Specific Form of Epidermal Growth Factor Receptor,”J. Virol., 2000, 74:9540-9545; Chu et al., “Toward Highly EfficientCell-Type-Specific Gene Transfer with Retroviral Vectors DisplayingSingle-Chain Antibodies,” J. Virol., 1997, 71:720-725; Kulkarni etal.,Programmed cell death signaling via cell-surface expression of asingle-chain antibody transgene,Transplantation Mar. 27,2000;69(6):1209-17.

[0538] IX.B. Non-Catalytic Derivatives of Active Sites of Enzymes

[0539] Enzymes bind their substrates, at least transiently, in regionsknown as “active sites.” It is known in the art that non-catalyticderivatives of enzymes, which bind but do not chemically alter theirsubstrates may be prepared. Non-catalytic enzymes, particularly themutant active sites thereof, are used to bind substrate molecules.

[0540] As a non-limiting example, enzymes from which biologicallyinactive (non-catalytic) sphingolipid-binding derivatives are obtained.Such derivatives of these enzymes bind their substrate sphingolipid.Sphingosine-1-phosphate (S1P) is bound by non-catalytic derivatives ofenzymes having S1P as a substrate, e.g., S1P lyase and S1P phosphatase.Sphingosine (SPH) is bound by non-catalytic derivatives of enzymeshaving SPH as a substrate, e.g., SPH kinase and ceramide synthase.Ceramide (CER) is bound by non-catalytic derivatives of enzymes havingCER as a substrate, such as, by way of non-limiting example, ceramidase,sphingomyelin synthase, ceramide kinase, and glucosylceramide synthase.Sphingomyelin is bound by non-catalytic derivatives of sphingomyelinase,an enzyme having sphingomyelin as a substrate.

[0541] IX.C. Nucleic Acid Binding Domains

[0542] Nucleic acid binding polypeptide domains may bind nucleic acidsin a sequence-dependent or sequence-independent fashion and/or in amanner that is specific for various nucleic acids having differentchemical structures (e.g., single- or double-stranded DNA or RNA,RNA:DNA hybrid molecules, etc.). Non-limiting examples of membrane-basedtranscription factors and DNA-binding protein include Smad proteins(Miyazono et al., TGF-beta signaling by Smad proteins (Review), AdvImmunol 75:115-57, 2000); SREBPs (sterol regulatory element bindingproteins) (Ye et al., Asparagine-proline sequence withinmembrane-spanning segment of SREBP triggers intramembrane cleavage bysite-2 protease, Proc Natl Acad Sci USA 97:5123-8, 2000; Shimomura etal., Cholesterol feeding reduces nuclear forms of sterol regulatoryelement binding proteins in hamster liver, Proc Natl Acad Sci USA94:12354-9, 1997; Brown and Goldstein, The SREBP pathway: regulation ofcholesterol metabolism by proteolysis of a membrane-bound transcriptionfactor (Review), Cell 89:331-40, 1997; Scheek et al., Sphingomyelindepletion in cultured cells blocks proteolysis of sterol regulatoryelement binding proteins at site 1, Proc Natl Acad Sci USA 94:11179-83,1997); mitochondrial DNA-binding membrane proteins, e.g., Abf2p andYhmZp (Cho et al., A novel DNA-binding protein bound to themitochondrial inner membrane restores the null mutation of mitochondrialhistone Abf2p in Saccharomyces cerevisiae, Mol Cell Biol 18:5712-23,1998); and bacterial DNA-binding membrane proteins (Smith et al.,Transformation in Bacillus subtilis: purification and partialcharacterization of a membrane-bound DNA-binding protein., J Bacteriol156:101-8, 1983).

[0543] IX.D. Attaching Binding Moities, or Other Compounds, to Minicells

[0544] Binding compounds or moieties can be chemically attached(conjugated) to minicells via membrane proteins that are displayed onthe minicells. The compound to be conjugated to minicells (the“attachable compound”) may of any chemical composition, i.e., a smallmolecule, a nucleic acid, a radioisotope, a lipid or a polypeptide. Onetype of attachable compound that can be covalently attached to minicellsis a binding moitiety, e.g., an antibody or antibody derivative. Anothernon-limiting example of attachable compounds is polyethylene glycol(“PEG”), which lowers the uptake in vivo of minicells by thereticuloendothelical system (RES). Another non-limiting example ofcreating stealth minicells to avoid the RES is to express proteins orother molecules on the surfaces of minicells whose lipid compositionshave been modified, such as anionic lipid-rich minicells.

[0545] By way of non-limiting example, it is possible to prepareminicells that express transmembrane proteins with cysteine moieties onextracellular domains. Linkage of the membrane protein may be achievedthrough surface cysteinyl groups by, e.g., reduction with cysteinylresidues on other compounds to form disulfide bridges (S═S). Ifappropriate cysteinyl residues are not present on the membrane proteinthey may be introduced by genetic manipulation. The substitution ofcysteine for another amino acid may be achieved by methods well-known tothose skilled in the art, for example, by using methods described inManiatis, Sambrook, and Fritsch (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1989). As a non-limiting example,bioactive lysosphingolipids (e.g., sphingosine, sphingosine-1-phosphate,sphingosylphosphoryl choline) are covalently linked to proteinsexpressed on the surfaces of minicells such that these bioactive lipidsare on the surface of the minicells and accessible for therapeutic ordiagnostic uses in vivo or in vitro.

[0546] When the attachable moiety and the membrane protein both have areduced sulfhydryl group, a homobifunctional cross-linker that containsmaleimide, pyridyl disulfide, or beta-alpha-haloacetyl groups may beused for cross-linking. Examples of such cross-linking reagents include,but are not limited to, bismaleimidohexane (BMH) or1,4-Di-[3′-(2′-pyridyldithio)propionamido]butane (DPDPB). Alternatively,a heterobifunctional cross-linker that contains a combination ofmaleimide, pyridyl disulfide, or beta-alpha-haloacetyl groups can beused for cross-linking.

[0547] As another non-limiting example, attachable moieties may bechemically conjugated using primary amines. In these instances, ahomobifunctional cross-linker that contains succiminide ester,imidoester, acylazide, or isocyanate groups may be used forcross-linking. Examples of such cross-linking reagents include, but arenot limited to: Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone(BSOCOES); Bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone(sulfo-BSCOCOES); Disuccinimidyl suberate (DSS);Bis-(Sulfosuccinimidyl)Suberate (BS3); Disuccinimidyl glutarate (DSG);Dithiobis(succinimidylpropionate) (DSP);Dithiobois(sulfosuccinimidylpropionate) (DTSSP); Disulfosuccinimidyltartrate (sulfo-DST); Dithio-bis-maleimidoethane (DTME); Disuccinimidyltartrate (DST); Ethylene glycolbis(sulfosuccinimidylsuccinate)(sulfo-EGS); Dimethyl malonimidate.2 HCl (DMM); Ethyleneglycolbis(succinimidylsuccinate) (EGS); Dimethyl succinimidate.2 HCl(DMSC); Dimethyl adipimidate.2 HCl (DMA); Dimethyl pimelimidate.2 HCl(DMP); and Dimethyl suberimlidate.2.HCl (DMS), and Dimethyl3,3′-dithiobispropionimidate.2 HCI (DTBP). Heterobifunctionalcross-linkers that contains a combination of imidoester or succinimideester groups may also be used for cross-linking.

[0548] As another non-limiting example, attachable moieties may bechemically conjugated using sulfhydryl and primary amine groups. Inthese instances, heterobifunctional cross-linking reagents arepreferable used. Examples of such cross-linking reagents include, butare not limited to: N-succinimidyl 3-(2- pyridyldithio)propionate(DPDP); N-succinimidyl 6-[3′-(2-pyridyldithio)-propionamido] hexanoate(sulfo-LC-SPDP); m-maleimidobenzoyl-N-hydoxysuccinimide ester (MBS);m-maleimidobenzoyl-N-hydoxysulfosuccinimide ester (sulfo-MBS);succinimidyl 4-[P-maleimidophenyl] butyrate (SMPB); sulfosuccinimidyl4-[p-maleimidophenyl] butyrate (sulfo-SMPB); N-[γ-Maleimidobutyryloxy]succinimide ester (GMBS), N-[γ-maleimidobutyryloxy] sulfosuccinimideester (sulfo-GMBS); N-[ε-maleimidocaproyloxy] succinimide ester (EMCS);N-[ε-maleimidocaproyloxy] sulfosuccinimide ester (sulfo-EMCS);N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB);sulfosuccinimidyl(4-iodacetyl)aminobenzoate (sulfo-SIAB); succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC); sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC);succiminidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amido-caproate)(LC-SMCC); 4-succinimidyloxycarbonyl-methyl-(2-pyridyldithio)toluene(SMPT); and sulfo-LC-SMPT.

[0549] As an exemplary protocol, a minicell suspension is made 5 mMEDTA/PBS, and a reducing solution of 2-mercaptoethylamine in 5 mMEDTA/PBS is added to the minicells. The mixture is incubated for 90minutes at 37° C. The minicells are washed with EDTA/PBS to removeexcess 2-mercaptoethylamine. The attachable moiety is dissolved in PBS,pH 7.2. A maleimide crosslinker is added to the solution, which is thenincubated for 1 hour at room temperature. Excess maleimide is removed bycolumn chromatography.

[0550] The minicells with reduced sulfhydryl groups are mixed with thederivatized compounds having an attachable moiety. The mixture isallowed to incubate at 4° C. for 2 hours or overnight to allow maximumcoupling. The conjugated minicells are washed to remove unreacted(unattached) compounds having the attachable moiety. Similar protocolsare used for expressed membrane proteins with other reactive groups(e.g., carboxyl, amine) that can be conjugated to an attachable moiety.

[0551] IX.E. Non-Genetic Methods for Directing Compounds to Membranes

[0552] Included within the scope of the invention are compounds that canbe inserted into the membrane of segregated minicells. Such compoundsinclude attachable moieties that are chemically conjugated to thesurface of a minicell, and compounds that associate with and/or insertinto a membrane “spontaneously,” i.e., by virtue of their chemicalnature. By way of non-limiting example, proteins that “spontaneously”insert into membranes include but are not limited to Thykaloid membraneproteins (Woolhead et al., J. Biol. Chem. 276:14607-14613, 2001), themitochondrial adenine nucleotide translocator (Jacotot et al., J. Exp.Med. 193:509-519, 2001), and polypeptides obtained using the methods ofHunt et al. (Spontaneous, pH-dependent membrane insertion of atransbilayer alpha-helix, Biochem 36:15177-15192, 1997). Lipids,gangliosides, sphingomyelins, plasmalogens glycosyl diacylglycerols, andsterols can also be incorporated into the membranes of segregatedminicells.

X. Membrane Proteins

[0553] In certain aspects of the invention, membrane proteins fromnon-eubacterial organisms are expressed and displayed by minicells. Thecellular membrane (a.k.a. the “plasma membrane”) is a lipid bilayer thatforms the boundary between the interior of a cell and its externalenvironment. The term “membrane proteins” refers to proteins that arefound in membranes including without limitation cellular and organellarmembranes.

[0554] X.A. Types of Membrane Proteins

[0555] X.A.1. In General

[0556] Membrane proteins consist, in general, of two types, peripheralmembrane proteins and integral membrane proteins.

[0557] Integral membrane proteins can span both layers (or “leaflets”)of a lipid bilayer. Thus, such proteins may have extracellular,transmembrane, and intracellular domains. Extracellular domains areexposed to the external environment of the cell, whereas intracellulardomains face the cytosol of the cell. The portion of an integralmembrane protein that traverses the membrane is the “transmembranedomain.” Transmembrane domains traverse the cell membrane often by oneor more regions comprising 15 to 25 hydrophobic amino acids which arepredicted to adopt an alpha-helical conformation.

[0558] Intergral membrane proteins are classified as bitopic orpolytopic (Singer, (1990) Annu. Rev. Cell Biol. 6:247-96). Bitopicproteins span the membrane once while polytopic proteins containmultiple membrane-spanning segments.

[0559] A peripheral membrane protein is a membrane protein that is boundto the surface of the membrane and is not integrated into thehydrophobic layer of a membrane region. Peripheral membrane proteins donot span the membrane but instead are bound to the surface of amembrane, one layer of the lipid bilayer that forms a membrane, or theextracellular domain of an integral membrane protein.

[0560] X.A.2. In General

[0561] The invention can be applied to any membrane protein, includingbut not limited to the following exemplary receptors and membraneproteins. The proteins include but are not limited to are receptors(e.g., GPCRs, sphingolipid receptors, neurotransmitter receptors,sensory receptors, growth factor receptors, hormone receptors, chemokinereceptors, cytokine receptors, immunological receptors, and complimentreceptors, FC receptors), channels (e.g., potassium channels, sodiumchannels, calcium channels.), pores (e.g., nuclear pore proteins, waterchannels), ion and other pumps (e.g., calcium pumps, proton pumps),exchangers (e.g., sodium/potassium exchangers, sodium/hydrogenexchangers, potassium/hydrogen exchangers), electron transport proteins(e.g., cytochrome oxidase), enzymes and kinases (e.g., protein kinases,ATPases, GTPases, phosphatases, proteases.), structural/linker proteins(e.g., Caveolins, clathrin), adapter proteins (e.g., TRAD, TRAP, FAN),chemotactic/adhesion proteins (e.g., ICAM11, selecting, CD34, VCAM-1,LFA-1, VLA-1), and phospholipases such as PI-specific PLC and otherphospholipiases.

[0562] X.A.3. Receptors

[0563] Within the scope of the invention are any receptor, includingwithout limitation:

[0564] The nuclear receptors, e.g the nuclear export receptor;

[0565] The peripheral (mitochondrial) benzodiazephine receptor (Gavishet al., “Enigma of the Peripheral Benzodiazephine Receptor,”Pharmacological Reviews, Vol.51, No.4);

[0566] Adrenergic and muscarinic receptors (Brodde et al., “Adrenergicand Muscarinic Receptors in the Human Heart”, Pharmacological Review,Vol. 51, No. 4);

[0567] Gamma-aminobutyric acid_(A) receptors (Barnard et al.,“International Union of Pharmacology. IV. Subtypes of γ-AminobutyricAcid Receptors: Classification on the Basis of Submit Structure andReceptor Function,” Pharmacological Reviews, Vol. 50, No. 2);

[0568] Kinin B₁ receptors (Marceau et al., “The B₁ Receptors forKinins,” Pharmacological Reviews, Vol. 50, No. 3);

[0569] Chemokine receptors (Murphy et al., “International Union ofPharmacology. XXII. Nomenclature for Chemokine Receptors”Pharmacological Reviewa, Vol. 52, No. 1);

[0570] Glycine and NMDA Receptors (Danysz et al., “Glycine andN-Methyl-D-Aspartate Receptors: Physiological Significance and PossibleTherapeutic Applications,” Pharmacological Reviews, Vol. 50, No. 4);

[0571] Glutamate receptor ion channels (Dingledine et al., “TheGlutamate Receptor Ion Channels”, Pharmacological Reviews, Vol. 51, No.1);

[0572] Purine and pyrimidine receptors including purinergic (e.g., P2)receptors (Ralevic et al., “Receptors for Purines and Pyrimidines”,Pharmacological Reviews, Vol. 50, No. 3); CNS receptors and membranetransporters (E. Sylvester Vizi, “Role of High-Affinity Receptors andMembrane Transporters in Nonsynaptic Communication and Drug Action inthe Central Nervous System,” Pharmacological Reviews, Vol. 52, No. 1);

[0573] Opoid receptors, including but not limited to the δ-opioidreceptor (Quock et al., “The δ-Opioid Receptor: Molecular Pharmacology,Signal Transduction and the Determination of Drug Efficacy”,Pharmacological Review, Col. 51, No. 3);

[0574] Angiotensin II receptors (Gasparo et al., “International Union ofPharmacology. XXIII. The Angiotensin II Receptors” Pharmalogical Review,Vol. 52, No. 3);

[0575] Cholecystokinin receptors (Noble et al., “International Union ofPharmacology. XXI. Structure, Distribution, and Functions ofCholecystokinin Receptors”, Pharmacological Reviews, Vol. 51, No.4)

[0576] Hormone receptors, including but not limited to, the estrogenreceptor; the glucocorticoid receptor; and the insulin receptor;

[0577] Receptors found predominantly in the central nervous system,including but not limited to, neuronal nicotinic acetylcholinereceptors; the dopamine D2/D3 receptor; GABA receptors; centralcannabinoid receptor CB1; opoid receptors, e.g., the kappa opioidreceptor, and the methadone-specific opioid receptor; nicotinicacetylcholine receptors; serotonin receptors, e.g., the serotonin 5-HT3receptor, the serotonin 5-HT4 receptor, and the serotonin-2 receptor;and dopamine receptors, e.g., the dopamine D2/D3 receptor; and theneurotensin receptor;

[0578] Receptors for growth factors, including but not limited to, theerythropoietin receptor; the FGF receptor; the EGF receptor; the VEGFreceptor; VEGF receptor-2 protein; VEGF-receptor protein (KDR);fibroblast growth factor receptor; the p75 nerve growth factor receptor;epidermal growth factor receptor; IGF-1 receptor; platelet factor-4receptor; alpha platelet-derived growth factor receptor; hepatocytegrowth factor receptor; and human fibroblast growth factor receptor;

[0579] Receptors for sphingolipids and lysophospholipids such as the Edgfamily of GPCRs;

[0580] Receptors for interleukins, e.g., receptors for interleukin-1(IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, et seq.; and

[0581] Various receptors, including by way of non-limiting example,receptors described in U.S. Pat. No. 6,210,967 (DNA encoding a mammalianLPA receptor and uses thereof); U.S. Pat. No. 6,210,921 (CAR: a novelcoxsackievirus and adenovirus receptor; U.S. Pat. No. 6,211,343(Lactoferrin receptor protein; U.S. Pat. No. 6,218,509 (LH/CG receptor,DNA and use thereof; U.S. Pat. No. 6,214,972 (DNA encoding prostaglandinreceptor DP); U.S. Pat. No. 6,221,613 (DNA encoding a human melaninconcentrating hormone receptor (MCH1) and uses thereof); U.S. Pat. No.6,221,660 (DNA encoding SNORF25 receptor); U.S. Pat. No. 6,225,080(Mu-subtype opioid receptor); U.S. Pat. No. 6,222,015 (Estrogenreceptor); U.S. Pat. No. 6,228,610 (Human metabotropic glutamatereceptor subtypes (hmR4, hmR6, hmR7) and related DNA compounds); U.S.Pat. No. 6,235,496 (Nucleic acid encoding mammalian mu opioid receptor);U.S. Pat. No. 6,258,556 (cDNA and genomic clones encoding human .mu.opiate receptor and the purified gene product); U.S. Pat. No. 6,245,531(Polynucleotide encoding insect ecdysone receptor); U.S. Pat. No.6,225,531 Glucan elicitor receptor, DNA molecule coding therefor,fungus-resistant plants transformed with the DNA molecule and method forcreating the plants); U.S. Pat. No. 6,245,893 (Receptor that bindsanti-convulsant compounds); U.S. Pat. No. 6,248,712 (Urokinase-typeplasminogen activator receptor; U.S. Pat. No. 6,248,554 (DNA sequencecoding for a BMP receptor); U.S. Pat. No. 6,248,520 (Nucleic acidmolecules encoding nuclear hormone receptor coactivators and usesthereof); U.S. Pat. No. 6,242,251 (Rhesus neuropeptide Y5 receptor);U.S. Pat. No. 6,252,056 (Human lysophosphatidic acid receptor and usethereof); U.S. Pat. No. 6,255,472 (Isolated nucleic acid moleculeencoding a human skeletal muscle-specific receptor); U.S. Pat. No.6,291,207 (Herpes virus entry receptor protein); U.S. Pat. No. 6,291,206(BMP receptor proteins); U.S. Pat. No. 6,291,195 (DNA encoding a humanmelanin concentrating hormone receptor (MCHII1) and uses thereof); U.S.Pat. No. 6,344,200 (Lactoferrin receptor protein); U.S. Pat. No.6,335,180 (Nucleic acid sequences encoding capsaicin receptor and usesthereof); U.S. Pat. No. 6,265,184 (Polynucleotides encoding chemokinereceptor 88C); U.S. Pat. No. 6,207,799 (Neuropeptide Y receptor Y5 andnucleic acid sequences); U.S. Pat. No. 6,290,970 (Transferrin receptorprotein of Moraxella); U.S. Pat. No. 6,326,350 (Transferrin receptorsubunit proteins of Neisseria meningitidis); U.S. Pat. No. 6,313,279(Human glutamate receptor and related DNA compounds); U.S. Pat. No.6,313,276 (Human endothelin receptor); U.S. Pat. No. 6,307,030 (Androgenreceptor proteins, recombinant DNA molecules coding for such, and use ofsuch compositions); U.S. Pat. No. 6,306,622 (cDNA encoding a BMP type IIreceptor); U.S. Pat. No. 6,300,087 (DNA encoding a human serotoninreceptor (5-HT4B) and uses thereof); U.S. Pat. No. 6,297,026 (Nucleicacids encoding the C140 receptor); U.S. Pat. No. 6,277,976 (Or-1, anorphan receptor belonging to the nuclear receptor family); U.S. Pat. No.6,274,708 (Mouse interleukin-11 receptor); U.S. Pat. No. 6,271,347(Eosinophil eotaxin receptor); U.S. Pat. No. 6,262,016 (Transferrinreceptor genes); U.S. Pat. No. 6,261,838 (Rat melanocortin receptorMC3-R); U.S. Pat. No. 6,258,943 (Human neurokinin-3 receptor); U.S. Pat.No. 6,284,870 (Gamma retinoic acid receptor); U.S. Pat. No. 6,258,944(OB receptor isoforms and nucleic acids encoding them); U.S. Pat. No.6,261,801 (Nucleic acids encoding tumor necrosis factor receptor 5);U.S. Pat. No. 6,261,800 (Luteinizing hormone/choriogonadotropin (LH/CG)receptor); U.S. Pat. No. 6,265,563 (Opioid receptor genes); U.S. Pat.No. 6,268,477 (Chemokine receptor 88-C); U.S. Pat. No. 6,316,611 (HumanN-methyl-D-aspartate receptor subunits, nucleic acids encoding same anduses therefor); U.S. Pat. No. 6,316,604 (Human C3b/C4b receptor (CR1));U.S. Pat. No. 6,287,855 (Nucleic acid encoding rat galanin receptor(GALR2)); U.S. Pat. No. 6,268,221 (Melanocyte stimulating hormonereceptor and uses); and U.S. Pat. No. 6,268,214 (Vectors encoding amodified low affinity nerve growth factor receptor).

[0582] X.A.3. Other Membrane Proteins

[0583] Other membrane proteins are within the scope of the invention andinclude but are not limited to channels (e.g., potassium channels,sodium channels, calcium channels.), pores (e.g., nuclear pore proteins,water channels), ion and other pumps (e.g., calcium pumps, protonpumps), exchangers (e.g., sodium/potassium exchangers, sodium/hydrogenexchangers, potassium/hydrogen exchangers), electron transport proteins(e.g., cytochrome oxidase), enzymes and kinases (e.g., protein kinases,ATPases, GTPases, phosphatases, proteases.), structural/linker proteins(e.g., Caveolins, clathrin), adapter proteins (e.g., TRAD, TRAP, FAN),

[0584] X.A.3.a. Cellular Adhesion Molecules

[0585] Cellular adhesion molecules, including but not limited to humanrhinovirus receptor (ICAM-1), ICAM-2, ICAM-3, and PECAM-1, andchemotactic/adhesion proteins (e.g., ,selectins, CD34, VCAM-1, LFA-1,VLA-1) are within the scope of the invention. See also Alpin et al.,“Signal Transduction and Signal Modulation by Cell Adhesion Receptors:The Role of Integrins, Cadherins, Immunoglobulin-Cell AdhesionMolecules, and Selectins”, Pharmacological Reviews, Vol. 50, No. 2.

[0586] X.A.3.b. Cytochrome P450 Enzymes

[0587] The family of enzymes known as “cytochrome P450” enzymes (sincethey absorb light in the 450 nanometer range), or as “cytochromeoxidase” enzymes (since they oxidize a wide range of compounds that donot naturally occur in circulating blood), are included within the scopeof the invention. P450 enzymes encompasses a variety of enzymes, many ofwhich are involved in xenobiotic metabolism, including by way ofnon-limiting example the metabolism of drugs, prodrugs and toxins.Directories and databases of P450s, and information regarding theirsubstrates, are available on-line (Fabian et al., The Directory ofP450-containing Systems in 1996, Nucleic Acids Research 25:274-277,1997). In humans, at least about 200 different P450s are present (for areview, see Hasler et al., Human cytochromes P450, Molecular Aspects ofMedicine 20:1-137, 1999). There are multiple forms of these P450s andeach of the individual forms exhibit degrees of specificity towardsindividual compounds or sets of compounds. In some cases, a substrate,whether it is a drug or a carcinogen, is metabolized by more than onecytochrome P450.

[0588] Members of the cytochrome P450 family are present in varyinglevels and their expression and activities are controlled by variablessuch as chemical environment, sex, developmental stage, nutrition andage. The cytochrome P450s are found at high concentrations in livercells, and at lower concentrations in other organs and tissues such asthe lungs (e.g., Fonne-Pfister et al., Xenobiotic and endobioticinhibitors of cytochrome P-450dbl function, the target of thedebrisoquine/sparteine type polymorphism, Biochem. Pharmacol.37:3829-35, 1988). By oxidizing lipophilic compounds, which makes themmore water-soluble, cytochrome oxidase enzymes help the body eliminate(via urine, or in aerosols exhaled out of the lungs) compounds thatmight otherwise act as toxins or accumulate to undesired levels.

[0589] In humans, several cytochrome P450s have been identified as beinginvolved in xenobiotic metabolism. These include CYP1A1, CYP1A2, CYP2A6,CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, andCYP3A5 (Crespi et al., The use of heterologous expressed drugmetabolizing enzymes-state of the art and prospects for the future,Pharm Ther 84:121-131, 1999).

[0590] X.A.3.c. Miscellaneous Membrane Proteins

[0591] In addition to the preceding non-limiting examples, the inventioncan be applied to the membrane proteins described in U.S. Pat. No.6,335,018 (High molecular weight major outer membrane protein ofmoraxella); U.S. Pat. No. 6,264,954 (Haemophilus outer membraneprotein); U.S. Pat. No. 6,197,543 (Human vesicle membrane protein-likeproteins); U.S. Pat. No. 6,121,427 (Major outer membrane protein CD ofbranhamella); U.S. Pat. Nos. 6,083,743 and 6,013,514 (Haemophilus outermembrane protein); U.S. Pat. No. 6,004,562 (Outer membrane protein B1 ofMoraxella catarrhalis); U.S. Pat. No. 5,863,764 (DNA encoding a humanmembrane protein); U.S. Pat. No. 5,861,283 (DNA encoding a limbicsystem-associated membrane protein); U.S. Pat. No. 5,824,321 (Clonedleptospira outer membrane protein); U.S. Pat. No. 5,821,085 (Nucleotidesequences of a T. pallidum rare outer membrane protein); U.S. Pat. No.5,821,055 (Chlamydia major outer membrane protein); U.S. Pat. No.5,808,024 (Nucleic acids encoding high molecular weight major outermembrane protein of moraxella); U.S. Pat. No. 5,770,714 (Chlamydia majorouter membrane protein); U.S. Pat. No. 5,763,589 (Human membraneprotein); U.S. Pat. No. 5,753,459 (Nucleotide sequences of T. pallidumrare outer membrane protein); U.S. Pat. No. 5,607,920 (Concanavalin abinding proteins and a 76 kD chondrocyte membrane protein (CMP) fromchondrocytes and methods for obtaining same); and U.S. Pat. No.5,503,992 (DNA encoding the 15kD outer membrane protein of Haemophilusinfluenzae).

[0592] X.B. Membrane Anchoring Domains

[0593] A membrane-anchoring domain can be incorporated into a fusionprotein of the invention. Non-limiting examples of membrane anchoringdomains include those derived from Prostaglandin H2 synthases (PGHS-1and -2) (Nina et al., Anchoring of a monotopic membrane protein: thebinding of prostaglandin H2 synthase-1 to the surface of a phospholipidbilayer, Eur. Biophys. J. 29:439-54, 2000; Otto and Smith, Photolabelingof prostaglandin endoperoxide H synthase-1 with3-trifluoro-3-(m-[1251]iodophenyl)diazirine as a probe of membraneassociation and the cyclooxygenase active site, J Biol Chem 271:9906-10,1996; and Otto and Smith, The orientation of prostaglandin endoperoxidesynthases-1 and -2 in the endoplasmic reticulum, J Biol Chem269:19868-75, 1994; those derived from carboxypeptidase E (EC 3.4.17.10)(Fricker et al., Identification of the pH-dependent membrane anchor ofcarboxypeptidase E (EC 3.4.17.10), J. Biol. Chem., 265, 2476-2482,1990); and peptide convertase 3 (PC3) (Smeekens et al., Identificationof a cDNA encoding a second putative prohormone convertase related toPC2 in AtT20 cells and islets of Langerhans, Proc Natl Acad Sci USA 88,340-344, 1990).

[0594] X.C. Transmembrane Domains

[0595] A variety of types and examples of transmembrane domain areknown. Proteins with up to 12 transmembrane domains are known (Fujiwaraet al., Identification of thyroid hormone transporters in humans:different molecules are involved in a tissue-specific manner,Endocrinology 2001 142:2005-12; Sharina et al., Mutational analysis ofthe functional role of conserved arginine and lysine residues intransmembrane domains of the murine reduced folate carrier, MolPharmacol 2001 59:1022-8). However, the invention is not limited to anyparticular number of transmembrane domains.

[0596] Monotropic (“single pass”) domains, which traverse a membraneonce, include by way of non-limiting example, those found in receptorsfor epidermal growth factor (EGF), receptors for tumor necrosis factor(TNF) and the like. Polytropic (“multipass”) proteins traverse amembrane two or more times. Non-limiting examples of polytropic proteinsare as follows.

[0597] Biotropic (“2 passes”) membrane proteins include, but are notlimited to: EnvZ of E. coli; the peroxisomal membrane protein Pex11-1p(Anton et al., ARF- and coatomer-mediated peroxisomal vesiculation, CellBiochem Biophys 2000;32 Spring:27-36); pleitropic drug ABC transportersof S. cervisiae (Rogers et al., The pleitropic drug ABC transportersfrom Saccharomyces cerevisiae, J Mol Microbiol Biotechnol 20013:207-14); and human and rate urate transporters hUAT and rUAT(Lipkowitz et al., Functional reconstitution, membrane targeting,genomic structure, and chromosomal localization of a human uratetransporter, J Clin Invest 2001 107:1103-15).

[0598] Tritropic (“3 pass”) membrane proteins include, but are notlimited to: the ethylene receptor ETR1 of Arabidopsis; the CauliflowerCard Expression protein CC 1 (Palmer et al., A Brassica oleracea GeneExpressed in a Variety-Specific Manner May Encode a Novel PlantTransmembrane Receptor, Plant Cell Physiol 2001 42:404-413); and asplice variant of the mitochondrial membrane protein hMRS3/4 (Li et al.,Characterization of a novel human putative mitochondrial transporterhomologous to the yeast mitochondrial RNA splicing proteins 3 and 4,FEBS Lett 2001 494:79-84).

[0599] Tetraspanins or tetraspans are non-limiting examples of membraneproteins with four transmembrane domains. (Levy et al., J. Biol. Chem,226:14597-14602, 1991; Tomlinson et al., J. Immol. 23:136-40, 1993; andBarclay et al., (In) The Leucocyte antigen factbooks, Academic press,London, 1993). These proteins are collectively known as the‘transmembrane 4 superfamily’ (TM4) because they span the plasmamembrane four times. The proteins known to belong to this familyinclude, but are not limited to: mammalian antigen CD9 (MIC3), a proteininvolved in platelet activation and aggregation; mammalian leukocyteantigen CD37, expressed on B lymphocytes; mammalian leukocyte antigenCD53 (OX-44), which may be involved in growth regulation inhematopoietic cells; mammalian lysosomal membrane protein CD63(Melanoma-associated antigen ME491; antigen AD1); mammalian antigen CD81(cell surface protein TAPA-1), which may play an important role in theregulation of lymphoma cell growth; mammalian antigen CD82 (Protein R2;Antigen C33; Kangai 1 (KAI1)), which associates with CD4 or CD8 anddelivers costimulatory signals for the TCR/CD3 pathway; mammalianantigen CD151 (SFA-1); Platelet-endothelial tetraspan antigen 3(PETA-3); mammalian TM4SF2 (Cell surface glycoprotein A15; TALLA-1;MXS1); mammalian TM4SF3 (Tumor-associated antigen CO-029); mammalianTM4SF6 (Tspan-6; TM4-D); mammalian TM4SF7 (Novel antigen 2 (NAG-2);Tspan-4); mammalian Tspan-2; Mammalian Tspan-3 (TM4-A); mammalianTetraspan NET-5; and Schistosoma mansoni and japonicum 23 Kd surfaceantigen (SM23/SJ23).

[0600] Non-limiting examples of membrane proteins with six transmembranedomains include the EBV integral membrane protein LMP-1, and a splicevariant of the mitochondrial protein hMRS3/4 (Li et al.,Characterization of a novel human putative mitochondrial transporterhomologous to the yeast mitochondrial RNA splicing proteins 3 and 4,FEBS Lett 2001 Apr 6;494(1-2):79-84). Proteins with six transmembranedomains also include STEAP (six transmembrane epithelial antigens of theprostate) proteins (Afar et al., U.S. Pat. No. 6,329,503). The prototypemember of the STEAP family, STEAP-1, appears to be a type IIIa membraneprotein expressed predominantly in prostate cells in normal humantissues. Structurally, STEAP-1 is a 339 amino acid protein characterizedby a molecular topology of six transmembrane domains and intracellularN- and C-termini, suggesting that it folds in a “serpentine” manner intothree extracellular and two intracellular loops.

[0601] Literally hundreds of 7-pass membrane proteins are known.G-protein coupled receptors (GPCRs), including without limitationbeta-adreno receptors, adrenergic receptors, EDG receptors, adenosinereceptors, B receptors for kinins, angiotensin receptors, and opiodreceptors are of particular interest. GPCRs are described in more detailelsewhere herein.

[0602] A non-limiting example of a protein with 9 transmembrane domainsis Lipocalin-1 interacting membrane receptor (Wojnar et al., Molecularcloning of a novel Lipocalin-1 interacting human cell membrane receptor(LIMR) using phage-display, J Biol Chem 2001 3; [epub ahead of print]).

[0603] Proteins with both transmembrane and anchoring domains are known.For example, AMPA receptor subunits have transmembrane domains and onemembrane-anchoring domain.

[0604] A variety of databases that describe known, and software programsthat predict, membrane anchoring and transmembrane domains are availableto those skilled in the art. See, for example Gcrdb.dba GCRDb [G ProteinCoupled Receptor database], Tmbase.dba Tmbase [database of transmembranedomains], Prodom.srv ProDom [Protein domains], Tmap.srv TMAP [Proteintransmembrane segments prediction], Tm7.srv TM7 [Retrieval of data on Gprotein-coupled receptors], and Memsat.sof MEMSAT [transmembranestructure prediction program].

[0605] Quentin and Fichant (J Mol Microbiol Biotechnol 2000 2:501-4,ABCdb: an ABC transporter database) have described a database devoted tothe ATP-binding cassette (ABC) protein domains (ABCdb), the majority ofwhich energize the transport of compounds across membranes. In bacteria,ABC transporters are involved in the uptake of a wide range of moleculesand in mechanisms of virulence and antibiotic resistance. In eukaryotes,most ABC transporters are involved in drug resistance, and many areassociated with diseases. ABCdb can be accessed via the World Wide Web(http://ir2lcb.cnrs-mrs.fr/ABCdb/). See also Sanchez-Fernandez et al.,The Arabidopsis thaliana ABC protein superfamily: a complete inventory,J Biol Chem May 9, 2001; [epub ahead of print], and Rogers et al., Thepleitropic drug ABC transporters from Saccharomyces cerevisiae, J MolMicrobiol Biotechnol Apr. 3, 2001 (2):207-14.

[0606] X.D. Functions and Activites of Membrane Proteins

[0607] Non-limiting examples of membrane proteins includemembrane-associated enzymes. Membrane-associated enzymes include but notlimited to certain enzymes of the electron transport chain (ETC),antigenic proteins such as the major histocompatability (MHC) antigens,transport proteins, channels, hormone receptors, cytokine receptors,glucose permeases, gap junction proteins and bacteriorhodopsins.

[0608] A “transport protein” or “transporter” is a type of membraneprotein that allows substances to cross plasma membranes at a rate thatis faster than what is found by diffusion alone. Some transport proteinsexpend energy to move substances (active transport). Many activetransport proteins are ATPases (e.g., the Na⁺-K⁺ATPase), or at leastbind ATP by virtue of comprising an ATP-binding cassette (ABC) (see,e.g., Rogers et al., The pleitropic drug ABC transporters fromSaccharomyces cerevisiae, J Mol Microbiol Biotechnol 3:207-14, 2001).Nucleobase transporters are reviewed by De Koning and Diallinas(Nucleobase Transporters, Mol Membr Biol 17:75-94, 2000).

[0609] A “channel protein” is a protein that facilitates the diffusionof molecules/ions across lipid membranes by forming a hydrophilic poreor “channel” that provides molecules/ions access through lipidmembranes, which are generally hydrophobic. Channels are oftenmultimeric, with the pore being formed by subunit-subunit interactions.

[0610] A “receptor” is a molecular entity, typically a protein, that isdisplayed on the surface of a cell. A receptor is characterized by highaffinity, often a specific binding of a specific substance, typicallyresulting in a specific biochemical or physiological effect.

[0611] A “hormone” is a naturally occurring substance secreted byspecialized cells that affects the metabolism or behavior of other cellshaving receptors for the hormone. Non-limiting examples of hormoneshaving receptors include but are not limited to insulin, cytokines,steroid hormones, histamines, glucagon, angiotensin, catecholamines, lowdensity lipids (LDLs), tumor necrosis factor alpha, tumor necrosisfactor beta, estrogen, and testosterone.

[0612] X.E. G-Protein-Coupled Receptors

[0613] G protein-coupled receptors (GPCRs) constitute the most prominentfamily of validated drug targets within biomedical research and arethought to be involved in such diseases and disorders as heart disease,hypertension, cancer, obesiy, and depression and other mental illnesses.Over half of approved drugs elicit their therapeutic effects byselectively addressing members of this target family and more than 1000sequences of the human genome encode for GPCRs containing the classical7-pass membrane structure characteristic of this family of proteins(Marinissen, M. and J. S. Gutkind, G-protien-coupled receptors andsignaling networks: emerging paradigms (Review), Trends. Phamacol. Sci.22: 368-376, 2001). Many pharmacological drug companies are interestedin the study of G-coupled proteins. It is possible to co-express aG-coupled protein receptor and its associated G-protein to study theirpharmacological characteristics (Strosberg and Marullo, Functionalexpression of receptors in microorganisms. TiPS, 1992. 13: 95-98).

[0614] G-protein-coupled receptors (GPCRs) are reviewed by Marinissen,M. and J. S. Gutkind, G-protien-coupled receptors and signalingnetworks: emerging paradigms. Trends. Phamacol. Sci. 22: 368-376, 2001;Sautel and Milligan, Molecular manipulation of G-protein-coupledreceptors: a new avenue into drug discovery, Curr Med Chem 2000 889-96;Hibert et al., This is not a G protein-coupled receptor, TrendsPharmacol Sci 1993, 14:7-12; Wilson et al., Orphan G-protein-coupledreceptors: the next generation of drug targets?, Br J Pharmacol 1998,125:1387-92; Roth et al., G protein-coupled receptor (GPCR) traffickingin the central nervous system: relevance for drugs of abuse, DrugAlcohol Depend 1998, 51:73-85; Ferguson and Caron, G protein-coupledreceptor adaptation mechanisms, Semin Cell Dev Biol 1998, 9:119-27;Wank, G protein-coupled receptors in gastrointestinal physiology. I. CCKreceptors: an exemplary family, Am J Physiol 1998, 274:G607-13; Rohrerand Kobilka , G protein-coupled receptors: functional and mechanisticinsights through altered gene expression. (Review), Physiol Rev 1998,78:35-52; and Larhammar et al., The receptor revolution—multiplicity ofG-protein-coupled receptors. (Review), Drug Des Discov 1993, 9:179-88.

[0615] GPCR localization and regulation has been studied usingGFP-comprising fusion proteins (Kallal and Benovic, Using greenfluorescent proteins to study G-protein-coupled receptor localizationand trafficking. (Review), Trends Pharmacol Sci 2000 21:175-80; andFerguson, Using green fluorescent protein to understand the mechanismsof G-protein-coupled receptor regulation. (Review), Braz J Med Biol Res1998, 31:1471-7); and by using chimeric GPCRs (Milligan and Rees,Chimaeric G alpha proteins: their potential use in drug discovery.(Review), Erratum in: Trends Pharmacol Sci Jun. 20, 1999; (6):252.

[0616] GPCRs belong to a superfamily of at least 6 families ofreceptors, the most important of which is the main family, A. Members ofthe membrane protein gene superfamily of GPCRs have been characterizedas having seven putative transmembrane domains. The transmembranedomains are believed to represent transmembrane alpha-helices connectedby extracellular or cytoplasmic loops. A functional G-protein is atrimer which consists of a variable alpha subunit coupled to much moretightly-associated and constant beta and gamma subunits, althoughG-protein independent actions have been postulated (Marinissen, M. andJ. S. Gutkind, G-protien-coupled receptors and signaling networks:emerging paradigms. Trends. Phamacol. Sci. 22: 368-376, 2001 Review). Avariety of ligands have been identified which function through GPCRs. Ingeneral, binding of an appropriate ligand (e.g., bioactive lipids, ions,bioactive amines, photons, odorants, hormones, neurotransmitters,peptides, nucleosides, etc.) to a GPCR leads to the activation of thereceptor. G-protein coupled receptors include a wide range ofbiologically active receptors, such as hormone, viral, growth factor andneuroreceptors. Typically, activation of a GPCR initiates the regulatorycycle of a corresponding G-protein. This cycle consists of GTP exchangefor GDP, dissociation of the alpha and beta/gamma subunits, activationof the second messenger pathway by a complex of GTP and the alphasubunit of the G-protein, and return to the resting state by GTPhydrolysis via the innate GTPase activity of the G-protein alpha subunitA.

[0617] GPCRs include, without limitation, dopamine receptors which bindto neuroleptic drugs used for treating psychotic and neurologicaldisorders. Other examples of members of this family include calcitonin,adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine,serotonin, histamine, thrombin, kinin, follicle stimulating hormone,opsins and rhodopsins, odorant, cytomegalovirus receptors, and the like.

[0618] Most GPCRs have single conserved cysteine residues in each of thefirst two extracellular loops which form disulfide bonds that arebelieved to stabilize functional protein structure. The seventransmembrane regions, each comprising conserved hydrophobic stretchesof about 20 to 30 amino acids, are designated as TM1, TM2, TM3, TM4,TM5, TM6, and TM7. TM3 is also implicated in signal transduction.

[0619] Although not wishing to be bound by any particular theory, it isbelieved that GPCRs participate in cell signaling through theirinteractions with heterotrimetric G-proteins composed of alpha, beta andgamma subunits (Marinissen, M. and J. S. Gutkind, G-protien-coupledreceptors and signaling networks: emerging paradigms. Trends. Phamacol.Sci. 22:368-376, 2001). In some aspects of the invention, GPCRs andhomologs are displayed on the surfaces of minicells.

[0620] X.F. EDG Receptors and Other Sphingolipid-Binding Receptors

[0621] The Endothelial Differentiation Gene (EDG) receptor familyincludes but is not limited to eight presently known GPCRs that have ahigh affinity to lipid ligands (Lynch et al., Life on the edg. TrendsPharmacol. Sci., 1999. 20: 273-5). These transmembrane receptors arefound in several different tissues in different species. EDG receptorshave been shown to be involved in calcium mobilization, activation ofmitogen-activated protein kinase, inhibition of adenylate cyclaseactivation, and alterations of the cytoskelaton. The EDG family isdivided into two different groups based on homology and ligandspecificity. The EDG 2, 4, and 7 receptors are specific for the ligandlysophosphatidic acid (LPA) (An et al., Signaling Mechanism andmolecular characteristics of G protein-coupled receptors forlysophosphatidic acid and sphingosine 1-phosphate. J. Cell Biochem,30/31:147-157, 1998; Goetzl et al., Distinctive expression and functionsof the type 4 endothelial differentiation gene-encoded G protein-coupledreceptor for lysophosphatidic acid in ovarian cancer. Cancer Res.,59:5370-5, 1999). In contrast, EDG 1, 3, and 5 bindsphingosine-1-phosphate (S1P) (Zhang et al., Comparative analysis ofthree murine G-protein coupled receptors activated bysphingosine-1-phosphate. Gene, 227:89-99, 1999). EDG -6 is believed tointeract with S1P (Yamazaki et al., Edg-6 as a putative sphingosine1-phosphate receptor coupling to Ca2⁺⁺ signaling pathway. Biochem PhysRes Com, 268:583-589, 2000).

[0622] Receptors that bind S1P and other sphingolipids are used in oneaspect of the invention (for a review of some S1P-binding receptors, seeSpiegel et al., Biochim. Biophys. Acta 1484:107-116, 2000). Suchreceptors include but are not limited to members of the EDG family ofreceptors (a.k.a. 1pA receptors, Chun, Crit. Rev. Neuro. 13:151-168,1999), and isoforms and homologs thereof such as NRG1 and AGR16.

[0623] EDG-1 was the first identified member of a class of Gprotein-coupled endothelial-derived receptors (EDG). Non-limitingexamples of other EDG family members that also bind S1P include EDG-3(a.k.a. ARG16; the rat homolog of EDG-3 is designated H218), EDG-5 ,EDG-6 and EDG-8. For reviews, see Goetzl et al., Adv. Exp. Med. Biol.469:259-264, 1999; and Chun et al., Cell. Biochem. Biophys. 30:213-242,1999).

[0624] EDG-1 is described by Lee et al., (Ann. NY Acad. Sci. 845:19-31,1998). Liu and Hla, The mouse gene for the inducible G-Protein-coupledreceptor edg-1. Genomics, 1997, 43: p.15-24. Human EDG-1c genes andproteins are described in published PCT application WO 99/46277 toBergsma et al.

[0625] EDG-3 is described by Okamoto et al. (Biochem. Biophys. Res.Commun. 260:203-208, 1999) and An et al. (FEBS Letts. 417:279-282,1997). See also An et al., J. Biol. Chem. 275:288-296, 2000.

[0626] EDG-5 human and mammalian genes are described in U.S. Pat. No.6,057,126 to Munroe et al. and published PCT application WO 99/33972 toMunroe et al. The rat homolog, H218, is described in U.S. Pat. No.5,585,476 to MacLennan et al. Van Brocklyn et al., J. Biol. Chem.274:4626-4632, 1999; and Gonda et al., Biochem. J. 337:67-75, 1999. Seealso An et al., J. Biol. Chem. 275:288-296, 2000.

[0627] EDG-6 is described by Graler et al. (Genomics 53:164-169, 1998),Yamazaki et al. (Biochem. Biophys. Res. Commun. 268:583-589, 2000), andVan Brocklyn et al. (Sphingosine-1-phosphate is a ligand for the Gprotein-coupled receptor EDG-6, Blood 95:2624-9, 2000).

[0628] EDG-8 from rat brain is described by Im et al., (J. Biol. Chem.275:14281-14286, 2000). Homologs of EDG-8 from other species, includinghumans, may also be used in the present invention.

[0629] The Mil receptor (Mil is an abbreviation for “miles apart”) bindsS1P and regulates cell migration during vertebrate heart development.The Mil receptor of Zebrafish is described by Mohler et al. (J. Immunol.151:1548-1561, 1993). Another S1P receptor is NRG1 (nerve growth factorregulated gene-1), the rat version of which has been identified(Glickman et al., Mol. Cel. Neurosci. 14:141-152, 1999).

[0630] Receptors that bind sphingosylphosphoryl choline (SPC) are alsoused in this aspect of the invention. Such receptors include but are notlimited to members of the SCaMPER family of receptors (Mao et al., Proc.Natl. Acad. Sci. U.S.A. 93:1993-1996, 1996; Betto et al., Biochem. J.322:327-333, 1997). Some evidence suggests that EDG-3 may bind SPC inaddition to S1P (Okamoto et al., Biochem. Biophys. Res. Commun.260:203-208, 1999). Derivatives of EDG-3 that bind both S1P and SPC areused in one aspect of the invention.

[0631] Receptors that bind lysophophatidic acid may be used in thepresent invention. These include EDG-2 (LPA1), EDG-4 (LPA2), EDG-7(LPA3). See Moller et al., Expression and function of lysophosphatidicacid receptors in cultured rodent microglial cells, J Biol Chem May 4,2001 [epub ahead of print]; Fukushima and Chun, The LPA receptors,Prostaglandins 64(1-4):21-32, 2001; Contos and Chun, The mouse1p(A3)/Edg7 lysophosphatidic acid receptor gene: genomic structure,chromosomal localization, and expression pattern, Gene 267:243-53, 2001;Schulte et al., Lysophosphatidic acid, a novel lipid growth factor forhuman thyroid cells: over-expression of the high-affinity receptor edg4in differentiated thyroid cancer, Int J Cancer 92249-56, 2001; Kimura etal., Two novel Xenopus homologs of mammalian LP(A1)/EDG-2 function aslysophosphatidic acid receptors in Xenopus oocytes and mammalian cells,J Biol Chem 276:15208-15, 2001; and Swarthout and Walling,Lysophosphatidic acid: receptors, signaling and survival (Review), CellMol Life Sci 57:1978-85, 2000.

[0632] Examples of lysophospholipid receptors including, but not limitedto EDG proteins, are disclosed in Fukushima et al. (Lysophospholipidreceptors. Annu. Rev. Pharmacol. Toxicol. 41:507-534, 2001) Malek andLee (Nrg-1 Belongs to the Endothelial Differentiation Gene Family of GProtein-coupled Sphingosine-1-phosphate Receptors, J. Biol. Chem.276:5692-5699, 2001), Hla et al. (Sphingosine-1-phosphate signaling viathe- EDG-1 family of G-protein-coupled receptors (Review), Ann N Y AcadSci 905:16-24, 2000; Chun, Lysophospholipid receptors: implications forneural signaling (Review), Crit Rev Neurobiol 13:151-68, 1999); and Chunet al. (A growing family of receptor genes for lysophosphatidic acid(LPA) and other lysophospholipids (LPs) (Review), Cell Biochem Biophys30:213-42, 1999).

XI. Recombinant DNA Expression

[0633] In order to achieve recombinant expression of a fusion protein,an expression cassette or construct capable of expressing a chimericreading frame is introduced into an appropriate host cell to generate anexpression system. The expression cassettes and constructs of theinvention may be introduced into a recipient eubacterial or eukaryoticcell either as a nonreplicating DNA or RNA molecule, which may be alinear molecule or, more preferably, a closed covalent circularmolecule. Since such molecules are incapable of autonomous replication,the expression of the gene may occur through the transient expression ofthe introduced sequence. Alternatively, permanent expression may occurthrough the integration of the introduced DNA sequence into the hostchromosome.

[0634] XI.A. Recombinant DNA Expression Systems

[0635] A variety of eubacterial recombinant DNA expression systems maybe used to produce the fusion proteins of the invention. Host cells thatmay be used in the expression systems of the present invention are notstrictly limited, provided that they are suitable for use in theexpression of the fusion protein of interest and can produce minicells.Non-limiting examples of recognized eubacterial hosts that may be usedin the present invention include bacteria such as E. coli, Bacillus,Streptomyces, Pseudomonas, Salmonella, Serratia, and the like.

[0636] Eubacterial expression systems utilize plasmid and viral(bacteriophage) expression vectors that contain replication sites andcontrol sequences derived from a species compatible with the host may beused. Suitable phage or bacteriophage vectors include λgt10, λgt11 andthe like. Suitable virus vectors may include pMAM-neo, pKRC and thelike. Appropriate eubacterial plasmid vectors include those capable ofreplication in E. coli (such as, by way of non-limiting example, pBR322,pUC118, pUC119, ColE1, pSC101, pACYC 184, πVX. See “Molecular Cloning: ALaboratory Manual” 1989). Bacillus plasmids include pC194, pC221, pT127,and the like (Gryczan, In: The Molecular Biology of the Bacilli,Academic Press, NY, pp. 307-329, 1982). Suitable Streptomyces plasmidsinclude p1J101 (Kendall et al., J. Bacteriol. 169:4177-4183, 1987), andStreptomyces bacteriophages such as C31 (Chater et al., In: SixthInternational Symposium on Actinomycetales Biology, Akademiai Kaido,Budapest, Hungary, pp. 45-54, 1986). Pseudomonas plasmids are reviewedby John et al. (Rev. Infect. Dis. 8:693-704, 1986), and Izaki (Jpn. J.Bacteriol. 33:729-742, 1978). See also Brent et al., “Vectors DerivedFrom Plasmids,” Section II, and Lech et al. “Vectors derived from Lambdaand Related Bacteriophages” Section III, in Chapter 1 of Short Protocolsin Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley andSons, New York, 1992, pages 1-13 to 1-27; Lech et al. “Vectors derivedfrom Lambda and Related Bacteriophages” Section III and Id. pages 1-28to page 1-52.

[0637] To express a protein, including but not limited to a fusionprotein, in a eubacterial cell, it is necessary to operably link the ORFencoding the protein to a functional eubacterial or viral promoter. Suchpromoters may be either constitutive or, more preferably, regulatable(i.e., inducible or derepressible). Examples of constitutive promotersinclude the int promoter of bacteriophage lambda, the bla promoter ofthe beta-lactamase gene sequence of pBR322, and the cat promoter of thechloramphenicol acetyl transferase gene sequence of pPR325, and thelike. Examples of inducible eubacterial promoters include the majorright and left promoters of bacteriophage lambda (P_(L) and P_(R)), thetrp, recA, lacZ, lac, and gal promoters of E. coli, the alpha-amylase(Ulmanen et al., J. Bacteriol. 162:176-182, 1985) and thesigma-28-specific promoters of B. subtilis (Gilman et al., Gene Sequence32:11-20, 1984), the promoters of the bacteriophages of Bacillus(Gryczan, in: The Molecular Biology of the Bacilli, Academic Press,Inc., NY, 1982), and Streptomyces promoters (Ward et al., Mol. Gen.Genet. 203:468-478, 1986). Eubacterial promoters are reviewed by Glick(Ind. Microbiot. 1:277-282, 1987), Cenatiempo (Biochimie 68:505-516,1986), and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).

[0638] Proper expression also requires the presence of aribosome-binding site upstream of the gene sequence-encoding sequence.Such ribosome-binding sites are disclosed, for example, by Gold et al.(Ann. Rev. Microbiol. 35:365-404, 1981). The selection of controlsequences, expression vectors, transformation methods, and the like, aredependent on the type of host cell used to express the gene. As usedherein, “cell”, “cell line”, and “cell culture” may be usedinterchangeably and all such designations include progeny. Thus, thewords “transformants” or “transformed cells” include the primary subjectcell and cultures derived therefrom, without regard to the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.However, as defined, mutant progeny have the same functionality as thatof the originally transformed cell.

[0639] Mammalian expression systems utilize host cells such as HeLacells, cells of fibroblast origin such as VERO or CHO-K1, or cells oflymphoid origin and their derivatives. Preferred mammalian host cellsinclude SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR332, which may provide better capacities for correct post-translationalprocessing. Non-limiting examples of mammalian extrachromosomalexpression vectors include pCR3.1 and pcDNA3.1, and derivatives thereofincluding but not limited to those that are described by and arecommercially available from Invitrogen (Carlsbad, Calif.).

[0640] Several expression vectors are available for the expression ofpolypeptides in mammalian host cells. A wide variety of transcriptionaland translational regulatory sequences may be employed, depending uponthe nature of the host. The transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, cytomegalovirus (CMV), simian virus, or the like, wherethe regulatory signals are associated with a particular gene sequencewhich has a high level of expression. Alternatively, promoters frommammalian expression products, such as actin, collagen, myosin, and thelike, may be employed. Transcriptional initiation regulatory signals maybe selected which allow for repression or activation, so that expressionof the gene sequences can be modulated. Of interest are regulatorysignals that are temperature-sensitive since, by varying thetemperature, expression can be repressed or initiated, or are subject tochemical (such as metabolite) regulation.

[0641] Preferred eukaryotic plasmids include, for example, BPV,vaccinia, SV40, 2-micron circle, and the like, or their derivatives.Such plasmids are well known in the art (Botstein et al., Miami Wntr.Symp. 19:265-274, 1982; Broach, in: The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p. 445-470, 1981; Broach, Cell28:203-204, 1982; Bollon et al., J. Clin. Hematol. Oncol. 10:39-48,1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, GeneSequence Expression, Academic Press, NY, pp. 563-608, 1980).

[0642] Expression of polypeptides in eukaryotic hosts generally involvesthe use of eukaryotic regulatory regions. Such regions will, in general,include a promoter region sufficient to direct the initiation of RNAsynthesis. Preferred eukaryotic promoters include, for example, thepromoter of the mouse metallothionein I gene sequence (Hamer et al., J.Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus(McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist etal., Nature (London) 290:304-31, 1981); and the yeast gal4 gene sequencepromoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975,1982; Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984).

[0643] Expression sequences and elements are also required for efficientexpression. Non-limiting examples include Kozak and IRES elements ineukaryotes, and Shine-Delgarno sequences in prokaryotes, which directthe initiation of translation (Kozak, Initiation of translation inprokaryotes and eukaryotes. Gene, 1999. 234: 187-208; Martinez-Salas etal., Functional interactions in internal translation initiation directedby viral and cellular IRES elements, Jour. of Gen. Virol. 82:973-984,2001); enhancer sequences; optioanl sites for repressor and inducers tobind; and recognition sites for enaymes that cleave DNA or RNA in asite-specific manner. Translation of mRNA is generally initiated at thecodon which encodes the first methionine; if so, it is preferable toensure that the linkage between a eukaryotic promoter and a preselectedORF does not contain any intervening codons that encode a methionine(i.e., AUG). The presence of such codons results either in the formationof a fusion protein with an uncharacterized N-terminal extension (if theAUG codon is in the same reading frame as the ORF) or a frame-shiftmutation (if the AUG codon is not in the same reading frame as the ORF).

[0644] XI. B. Expression of Membrane Proteins

[0645] Presently, the most commonly used expression systems for theexpression of integral membrane proteins are eukaryotic and eubacterialwhole cell expression systems. Although minicells have been used toexpress several eubacterial membrane proteins, the production ofnon-eubacterial membrane proteins has not been reported. One aspect ofthe invention is the discovery that the minicell expression system canbe made to express and preferably display integral membrane proteinsfrom non-eubacterial organisms.

[0646] Some commonly used expression systems include in vitro systems,such as the Rabbit Reticulocyte Lysate System and E. coli S30 ExtractSystem (both available from Promega) (Zubay, Methods Enz. 65:856, 1980)and in vivo systems, such as eukaryotic cell culture expression, andbacterial expression systems. Although this is not an exhaustive list,these systems are representative.

[0647] The Rabbit Reticulocyte Lysate system utilizes a cell lysate thatcontains all the enzymes required for transcription and translation todrive protein expression, and is a good in vitro system for producingsmall amounts of labeled and unlabeled protein. However, this system isnot well-suited for the production of large quantities of proteins andis limited to soluble proteins as there are no membranes in which toincorporate membrane proteins.

[0648] In eukaryotic cell culture systems, expression vectors suited forexpression in host eukaryotic cells are transfected into cultured cellsand protein is translated from mRNA produced from the vector DNAtemplate Kaufman, Overview of vector design for mammalian geneexpression. Mol Biotechnol, 2001. 16: 151-160; Lee, et al., Heterologousgene expression in avian cells: Potential as a producer of recombinantproteins. J Biomed Sci, 1999. 6: 8-17; Voorma et al., Initiation ofprotein synthesis in eukaryotes. Mol Biol Rep, 1994. 19: 139-45). Cellscan then either be harvested to prepare at least partially purifiedproteins or proteins produced from the expression element can be studiedin the host cell environment.

[0649] Regarding membrane proteins, such systems have limitations.Primary cell lines are difficult to maintain and are short lived.Immortalized cell lines divide indefinitely, but have been altered inmany ways and can be unpredictable. The transfection efficiency is verylow in most eukaryotic cells and some cell types are refractory totransformation. Moreover, other proteins are expressed in these cellsalong with the protein of interest. This can cause difficulties whenperforming certain experiments and when attempting to immunoprecipitatethe protein. Good experimental data are difficult to obtain from studiessuch as binding assays (because of high background due to endogenousproteins), and crystal determination of protein structure (because it isdifficult to obtain enough purified protein to efficiently formcrystals).

[0650] Bacterial expression systems are generally similar to that of theeukaryotic expression systems in that they both use the host cellenzymes to drive protein expression from recombinant expression vectors(Cornelis, P., Expressing genes in different Escherichia colicompartments. Curr Opin Biotechnol, 2000. 11: p. 450-454; Laage andLangosch, Strategies for prokaryotic expression of eukaryotic membraneproteins. Traffic, 2001. 2: 99-104; Pines, O. and M. Inouye, Expressionand secretion in E. coli. Mol Biotechnol, 1999. 12: 25-34).

[0651] In bacterial expression systems, bacterial cells are transformedwith expression elements, and transcription and translation is drivenfrom a bacterial promoter. Bacteria divide very rapidly and are easy toculture; it is relatively easy to produce a large number of bacteria ina short time. Moreover, incorporation of expression elements vector intobacterial cells is efficient. Transformed cells can be isolated thatarise from a single bacterium. Cultures of transformed cells are thusgenetically identical and all cells in the culture will contain theexpression element. However, there are proteins that are not suitablefor expression in bacteria because of differences between eukaryoticcells and bacterial cells in transcription, translation, andpost-translational modification.

[0652] The E. coli whole cell expression system has been used to expressfunctional integral membrane proteins. For a review, see Strosberg,Functional expression of receptors in microorganisms. TiPS, 1992. 13:95-98. Examples of mammalian integral membrane proteins that have beenexpressed in Escherichia coli include rat alpha-2B-adrenoceptors (Xia etal., Functional expression of rat β2B-adrenoceptor in E. coli. Euro J.Pharma, 1993. 246: 129-133) and the human beta2-adrenergic receptor(Marullo et al., IIuman β2adrenergic receptors expressed in Escherichiacoli membranes retain their pharmacological properties. Proc. Natl.Acad. Sci. USA, 1988. 85: 7551-7555). In some of these studies, theintegral membrane proteins were not only expressed in E. coli expressionsystems, but also retained their pharmacological properties. This allowsfor binding studies to be performed with minimal background signal(“noise”) from host cell proteins. It has also been shown that signalsequences (the short hydrophobic amino acid sequence at the N-terminusof integral membrane proteins that signals the transport of the proteinto the membrane) from mammalian cells may be functional in the E. colisystem.

[0653] As is discussed herein, the expression of membrane proteins suchas GPCRs, ion channels, and immuno-receptors in minicells, and theirincorporation into the membranes thereof, allows for the study and useof such non-eubacterial membrane proteins. The minicell system of theinvention is particularly well-suited for the study and expression ofEDG proteins because of the lipid nature of the ligands for thesereceptors. The identification of ligand binding kinetics andbiochemistry of these receptors because of the physiochemical propertiesof the lipid ligands (LPA and S1P), which results in high non-specificbinding (Lee et al., Sphingosine-1-phosphate is a ligand for the Gprotein-coupled receptor EDG-1. Science, 1998. 279: 1552-1555; VanBrocklyn et al., Sphingosine-1-phosphate is a ligand for the Gprotein-coupled receptor EDG-6. Blood, 2000. 95: 2624-2629; Liu et al.,Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, isessential for vascular maturation. J. Clin. Investigation, 2000. 106:951-961).

[0654] It is believed, for example, that in the case of the ionchannels, the minicell expression system is less cumbersome thenprocedures that are presently used to study properties of ion channels,such as, e.g., reconstitution studies (Montal, Molecular anatomy andmolecular design of channel proteins. FASEB J., 1990. 4: p. 2623-2635).Ionic conditions both inside and outside of minicells can be manipulatedin various ways, and the properties of an ion channel that is expressedin a minicell, and factors that activate or modulate the activities ofthe channel, can be studied. Binding and kinetic studies are performedon ligand mediated ion channels. This type of study is enhanced when theion channel is able to interact specifically with its ligand and has alow background of non-specific binding from the endogenous proteins.This can be accomplished by making the minicells into protoplasts orporoplasts in which the ligand-activated ion channels in the innermembrane are exposed to the external environment and have better accessto their specific ligand.

[0655] A “recombinant expression system” (or simply “expression system”)is one that directs the production of exogenous gene products in a hostcell or minicell of choice. By “expressed” it is meant that a geneproduct of interest (which can be a protein or nucleic acid) is producedin the expression system of choice.

[0656] Host cells (and/or minicells) harboring an expression constructare components of expression systems. An “expression vector” is anartificial nucleic acid molecule into which an exogenous ORF encoding aprotein, or a template of a bioactive nucleic acid can be inserted insuch a manner so as to be operably linked to appropriate expressionsequences that direct the expression of the exogenous gene. By the term“operably linked” it is meant that the part of a gene that istranscribed is correctly aligned and positioned with respect toexpression sequences that promote, are needed for and/or regulate thistranscription. The term “gene product” refers to either a nucleic acid(the product of transcription, reverse transcription, or replication) ora polypeptide (the product of translation) that is produced using thenon-vector nucleic acid sequences as a template.

[0657] In some applications, it is preferable to use an expressionconstruct that is an episomal element. If the episomal expressionconstruct expresses (or, preferably in some applications,over-expresses) a an ORF that has been incorporated into the episomalexpression construct, the minicells will direct the production of thepolypeptide encoded by the ORF. At the same time, any mRNA moleculestranscribed from a chromosomal gene prior to minicell formation thathave been transferred to the minicell are degraded by endogenous RNaseswithout being replaced by new transcription from the (absent) bacterialchromosome.

[0658] Chromosomally-encoded mRNAs will not be produced in minicells andwill be “diluted” as increasing amounts of mRNAs transcribed from theepisomal element are generated. A similar dilution effect is expected toincrease the relative amount of episomally-generated proteins relativeto any chromosomally-encoded proteins present in the minicells. It isthus possible to generate minicells that are enriched for proteinsencoded by and expressed from episomal expression constructs.

[0659] Although by no means exhaustive, a list of episomal expressionvectors that have been expressed in eubacterial minicells is presentedin Table 4.

[0660] It is also possible to transform minicells with exogenous DNAafter they have been prepared or separated from their parent cells. Forexample, phage RNA is produced in minicells after infection by lambdaphage (Witkiewicz and Taylor, Ribonucleic acid synthesis afteradsorption of the bacteriophage lambda on Escherichia coli minicells,Acta Microbiol Pol A 7:21-4, 1975), even though replication of lambdaphage may not occur in minicells (Witkiewicz and Taylor, The fate ofphage lambda DNA in lambda-infected minicells, Biochim Biophys Acta564:31-6, 1979).

[0661] Because it is the most characterized minicell-producing species,many of these episomal elements have been examined in minicells derivedfrom E. coli. It is understood by practitioners of the art, however,that many episomal elements that are expressed in E. coli also functionin other eubacterial species, and that episomal expression elements forminicell systems in other species are available for use in the inventiondisclosed herein.

[0662] In one aspect of the invention, eukaryotic and archeabacterialminicells are used for expression of membrane proteins, particularly ininstances where such desirable proteins have enhanced or alteredactivity after they undergo post-translational modification processessuch as phosphorlyation, proteolysis, mystrilation, GPI anchoring andglycosylation. Expression elements comprising expression sequenceoperably linked to ORFs encoding the membrane proteins of interest aretransformed into eukaryotic cells according to methods and usingexpression vectors known in the art. By way of non-limiting example,primary cultures of rat cardiomyocytes have been used to produceexogenous proteins after transfection of expression elements therefor byelectroporation (Nakajima et al., Expression and characterization ofEdg-1 receptors in rat cardiomyocytes: Calcium deregulation in responseto sphingosine-1-phosphate, Eur. J. Biochem. 267: 5679-5686, 2000).

[0663] Yeast cells that produce minicells are transformed withexpression elements comprising an ORF encoding a membrane proteinoperably linked to yeast expression sequences. Cells that harbor atransferred expression element may be selected using a gene that is partof the expression element that confers resistant to an antibiotic, e.g.,neomycin.

[0664] Alternatively, in one aspect of the invention, bacterialminicells are prepared that contain expression elements that areprepared from shuttle vectors. A “shuttle vector” has sequences requiredfor its replication and maintenance in cells from two different speciesof organisms, as well as expression elements, at least one of which isfunctional in bacterial cells, and at least one of which is functionalin yeast cells. For example, E. coli-yeast shuttle vectors are known inthe art and include, by way of non-limiting example, those derived fromYip, Yrp, Ycp and Yep. Preferred E. coli-yeast shuttle vectors areepisomal elements that can segregrate into yeast minicells (i.e., Yrp,Ycp and Yep. Particularly preferred are expression vectors of the Yep(yeast episomal plasmid) class, and other derivatives of the naturallyoccurring yeast plasmid known as the 2 μm circle. The latter vectorshave relatively high transformation frequencies and are stablymaintained through mitosis and melosis in high copy number. TABLE 4Episomal Elements That Segregate Into Escherichia coli MinicellsEPISOMAL ELEMENT REFERENCES Plasmids R6K, R1DRD19 Nesvera et al., FoliaMicrobiol. (Praha) 23: 278-285 (1978) PSC101 Fox et al., Blood 69:1394-1400 (1987) PBR322 Fox et al., Blood 69: 1394-1400 (1987) F elementCohen et al., Proc. Natl. Acad. Sci. 61: 61-68 (1968); Khachatourians G.G., Biochim. Biophys. Acta. 561: 294-300 (1979) NR1 Hochmannova et al.,Folia Microbiol. (Praha) 26: 270-276 R6δ1 Hochmannova et al., FoliaMicrobiol. (Praha) 26: 270-276 PTTQ18 Rigg et al., Arch. Oral. Biol. 45:41-52 (2000) PGPR2.1 Rigg et al., Arch. Oral. Biol. 45: 41-52 (2000);expresses cell surface antigen of P. gingivalis “mini-plasmid”derivative of Firshein et al., J. Bacteriol. 150: 1234-1243 (1982) RK2ColE1 Rashtchian et al., J. Bacteriol. 165: 82-87 (1986); Witkiewicz etal., Acta. Microbiol. Pol. A 7: 21-24 (1975) PSC101 Rashtchian et al.,J. Bacteriol. 165: 82-87 (1986); Curtiss, Roy, III, U.S. Pat. No.4,190,495; Issued Feb. 26, 1980 pACYC184 Chang et al., J. Bacteriol.134: 1141-1156 (1978); Rose, Nucleic Acids Res 16: 355 (1988) Co1Ib,Co1Ib7 DRD& Skorupska et al., Acta. Microbiol. Pol. A 8: 17-26 (1976)pUC19 Heighway et al., Nucleic Acids Res. 17: 6893-6901 (1989) R-plasmidHochmannova et al., Folia Microbiol. (Praha) 25: 11-15 (1980) PCR1Hollenberg et al., Gene 1: 33-47 (1976); yeast shuttle vectorBacteriophage Lambda Witkiewicz et al., Acta. Microbiol. Pol. A 7: 21-24(1975) M13 Staudenbauer et al., Mol. Gen. Genet. 138: 203-212 (1975) T7Libby, Mech Ageing Dev. 27: 197-206 (1984) P1 Curtiss, Roy, III, U.S.Pat. No. 4,190,495; Issued Feb. 26, 1980; J Bacteriol 1995; 177: 2381-6,Partition of P1 plasmids in Escherichia coli mukB chromosomal partitionmutants, Funnell and Gagnier.

[0665] For expression of membrane proteins, and/or other proteins ofinterest in the recipient cell, ORFs encoding such proteins are operablylinked to eukaryotic expression sequences that are appropriate for therecipient cell. For example, in the case of E. coli-yeast shuttlevectors, the ORFs are operably linked to expression sequences thatfunction in yeast cells and/or minicells. In order to assess theeffectiveness of a gene delivery vehicle, or a gene therapy expressionelement, an ORF encoding a detectable polypeptide (e.g., GFP,beta-galactosidase) is used. Because the detectable polypeptide isoperably linked to eukaryotic expression elements, it is not expressedunless it has been transferred to its recipient (eukaryotic) cell. Thesignal from the detectable polypeptide thus correlates with theefficiency of gene transfer by a gene delivery agent, or the degree ofexpression of a eukaryotic expression element.

[0666] Gyuris and Duda (High-efficiency transformation of Saccharoymcescells by bacterial minicell protoplast fusion, Mol Cel Biol 6:329507,1986) allegedly demonstrated the transfer of plasmid molecular by fusingminicell protoplasts with yeast protoplasts. Gyuris and Duda state that10% of Saccharomyces cerevisiae cells were found to contain transformingDNA sequences. However, the plasmids did not contain eukaryoticexpression elements, were not shuttle vectors, and genetic expression ofthe plasmids in yeast cells was not examined.

XII. Uses of Minicells in Research

[0667] XII.A. In General

[0668] The minicells of the invention can be used in researchapplications such as, by way of non-limiting example, proteomics,physiology, chemistry, molecular biology, physics, genetics, immunology,microbiology, proteomics, virology, pathology, botany, and neurobiology.Research applications include but are not limited to protein-ligandbinding studies, competitive inhibition studies, structural studies,protein interaction studies, transfection, signaling studies, viralinteraction studies, ELISA, antibody studies, gel electrophoresis,nucleotide acid) applications, peptide production, cell cultureapplications, cell transport studies, isolation and separation studies,chromatography, labeling studies, synthesis of chemicals, chemical crosslinking, flow cytometry, nanotechnology, micro switches, micro-machines,agricultural studies, cell death studies, cell-cell interactions,proliferation studies, and protein-drug interactions. Minicells areapplicable to research applications involving, by way of non-limitingexample, the elucidation, manipulation, production, replication,structure, modeling, observations, and characterization of proteins.

[0669] The types of proteins that can be involved in researchapplications of minicells can be either soluble proteins or membranebound proteins, and include but are not limited to receptors (e.g.,GPCRs, sphingolipid receptors, neurotransmitter receptors, sensoryreceptors, growth factor receptors, hormone receptors, chemokinereceptors, cytokine receptors, immunological receptors, and complimentreceptors, FC receptors), channels (e.g., potassium channels, sodiumchannels, calcium channels.), pores (e.g., nuclear pore proteins, waterchannels), ion and other pumps (e.g., calcium pumps, proton pumps),exchangers (e.g., sodium/potassium exchangers, sodium/hydrogenexchangers, potassium/hydrogen exchangers), electron transport proteins(e.g., cytochrome oxidase), enzymes and kinases (e.g., protein kinases,ATPases, GTPases, phosphatases, proteases.), structural/linker proteins(e.g., Caveolins, clathrin), adapter proteins (e.g., TRAD, TRAP, FAN),chemotactic/adhesion proteins (e.g., ICAM11, selecting, CD34, VCAM-1,LFA-1,VLA-1), and chimeric/fusion proteins (e.g., proteins in which anormally soluble protein is attached to a transmembrane region ofanother protein).

[0670] Research products are designed for any specific type ofapplication. These products may be packaged and distributed as, by wayof non-limiting example, kits, chemicals, solutions, buffers, powders,solids, filters, columns, gels, matrixes, emulsions, pellets, capsules,and aerosols. Kits and reagents for certain research applications may berequired by regulatory agency to be labeled “research use only” in orderto indicate that the reagents are not intended for use in humans.

[0671] XII.B. Transfection

[0672] Transfection is the process of introducing genetic material intoeukaryotic and archaebacterial cells using biological, biochemical orphysical methods. This process allows researchers to express and studytarget proteins in cultured cells (research use) as well as to delivergenetic material to cells in vivo or ex vivo systems (gene therapy).There are a variety of techniques which allow for the introduction andexpression of proteins into target cells. These include mechanicaltransfection (Biolistic particles and Electroporation), calciumphosphate, DEAE-dextran/polybrene, viral based techniques and lipidbased techniques.

[0673] The genetic material and/or nucleic acid to be delivered can be,by way of non-limiting example, nucleic acids that repair damaged ormissing genes, nucleic acids for research applications, nucleic acidsthat kill a dysfunctional cell such as a cancer cell, antisenseoligonucleotides to reduce or inhibit expression of a gene product,genetic material that increases expression of another gene, nucleotidesand nucleotide analogs, peptide nucleic acids (PNAs), tRNAs, rRNAs,catalytic RNAs, RNA:DNA hybrid molecules, and combinations thereof.

[0674] The genetic material may comprise a gene expressing a protein.exemplary proteins include, but are not limited to, receptors (e.g.,GPCRs, sphingolipid receptors, neurotransmitter receptors, sensoryreceptors, growth factor receptors, hormone receptors, chemokinereceptors, cytokine receptors, immunological receptors, and complimentreceptors, FC receptors), channels (e.g., potassium channels, sodiumchannels, calcium channels.), pores (e.g., nuclear pore proteins, waterchannels), ion and other pumps (e.g., calcium pumps, proton pumps),exchangers (e.g., sodium/potassium exchangers, sodium/hydrogenexchangers, potassium/hydrogen exchangers), electron transport proteins(e.g., cytochrome oxidase), enzymes and kinases (e.g., protein kinases,ATPases, GTPases, phosphatases, proteases), structural/linker proteins(e.g., Caveolins, clathrin), adapter proteins (e.g., TRAD, TRAP, FAN),chemotactic/adhesion proteins (e.g., ICAMI 11, selectins, CD34, VCAM-1,LFA-1, VLA-1), and chimeric/fusion proteins (e.g., proteins in which anormally soluble protein is attached to a transmembrane region ofanother protein).

[0675] A minicell that is used to deliver therapeutic agents maycomprise and display a binding moiety. By way of non-limiting example,binding moieties used for particular purposes may be a binding moietydirected to a compound or moiety displayed by a specific cell type orcells found predominantly in one type of tissue, which may be used,among other things, to target minicells and their contents to specificcell types or tissues. A preferred binding moiety is an antibody orantibody derivative. Other binding moieties include, but are not limitedto, receptors, enzymes, ligands, binding peptides, fusion proteins,small molecules conjugated to transmembrane proteins, ligands conjugatedto transmembrane proteins, viral fusion proteins, and fusion/chimericproteins.

[0676] A minicell containing genetic material may be to a target cell bymethods including, but not limited to, receptor mediated endocytosis,cell fusion, or phagocytosis (Aderem et al., Mechanism of Phagocytosisin Macrophages, Annu. Rev. Immunol. 17:593-623, 1999). The minicell genedelivery system is used to deliver genetic material in culture forresearch applications as well as to cells in vivo as part of genetherapy or other therapeutic applications.

[0677] By way of non-limiting example, a minicell may express a proteinsuch as invasin to induce receptor mediated endocytosis (Pepe et al.,“Yersinia enterocolitica invasin: A primary role in the initiation ofinfection,” Proc. Natl. Acad. Sci. U.S.A. 90:6473-6477, 1993; Alrutz etal., “Involvement of focal adhesion kinase in invasin-mediated uptake,”Proc. Natl. Acad. Sci. U.S.A. 95:13658-13663, 1998). Invasin interactswith the Beta2 Integrin protein and causes it to dimerize. Upondimerization the Beta2 Integrin signals for an endocytotic event. Thus aminicell expressing the invasin protein will be taken up by cellsexpressing Beta2 Integrin via endocytosis.

[0678] Another non-limiting example of the minicell gene delivery andtransfection system using invasin involves the expression of invasinfollowing a targeting event. In this example, a minicell expresses atargeting protein that is capable of bringing the minicell in contactwith a specific target cell. Upon contact with the target cell, theminicell will be induced to transcribe and translate invasin. Theinduction is accomplished via signaling events or with a transcriptionfactor dimerization event. The minicells can be engineered to containtargeting proteins that induce protein expression only upon contact witha specific target cell. By way of non-limiting example, the invasin isexpressed only at the target cell where it induces endocytosis, thuspreventing the minicell from entering any cell but the target cell.

[0679] Proteins can be induced and expressed post contact with targetcells include but are not limited to antibodies and antibodyderivatives, receptors, enzymes, ligands, binding peptides, fusionproteins, small molecules conjugated to transmembrane proteins, ligandsconjugated to transmembrane proteins, viral fusion proteins,antibiotics, apoptotic proteins, hormones, toxins, poisons, andfusion/chimeric proteins.

[0680] Another non-limiting example of gene delivery or transfectionusing the minicell involves the use of the type III secretion apparatusof bacteria. The type III secretion apparatus is expressed in theminicell and used to transfer genetic material to a target cell.

[0681] Another non-limiting example of gene delivery and transfectionusing minicells involves minicells that have been engineered to containanionic lipids or cationic lipids (Axel et al., “Toxicity, UptakeKinetics and Efficacy of New Transfection Reagents: Increase ofOligonucleotide Uptake,” Jour. of Vasc. Res. 040:1-14, 2000). Many typesof lipids have been shown to induce or enhance transfection and genedelivery in a variety of cell types. Minicells containing such lipidscould be used to transfer genetic material to specific cell types.Minicells can also be engineered to express targeting proteins thatwould allow the minicell to associate tightly with a target cell, whichwill facilitate the lipid interactions and gene transfer.

[0682] Another non-limiting example of gene delivery or transfectionusing minicells involves the use of ligands to induce receptor mediatedendocytosis. By way of non-limiting example, the ligand is expressed onthe surface of the minicell, or is attached to the surface of theminicell. A minicell containing genetic material is then able toassociate with a target cell expressing the target receptor for theligand. The receptor/ligand interaction will result in the endocytosisof the minicell into the target cell where the minicell would releaseand deliver the genetic material.

[0683] Another non-limiting example of gene delivery or transfectionusing minicells involves the use of fusion proteins, such as but notlimited to viral capsid proteins. In this example the fusion proteinwould be expressed or attached to the outside of the minicell. Thefusion protein would then induce fusion of a target cell with theminicell upon contact. The contact could be initiated via randomnon-targeting events or via the use of specific targeting proteins. Inboth cases the end result would be the fusion of the minicell with atarget cell and the delivery of the genetic material.

[0684] XII.C. Non-Limiting Examples of Research Applications ofMinicells

[0685] XII.C.1. Phage Interactions With Bacterial Membranes

[0686] One non-limiting example of a research application for minicellswould be the study of phage interactions with a bacterial membrane. Theminicells could be used to study how phage associate and enter into ahost bacterium. Another non-limiting example is the research applicationof minicells is to study isolated cell signaling pathways. The proteinsof a signaling pathway could be expressed in the minicell and the signalcascade could be monitored. Another non-limiting example of researchapplications is the use of minicells to determine how recombinationevents occur. In this example the minicell is used to provide anenvironment to study the recombination event between two episomalplasmid DNA units.

[0687] XII.C.2. Matrices

[0688] Another non-limiting example of a research application ofminicells is to form chromatography matrices for immunoprecipitation,isolation and separation techniques. The minicell can express anddisplay target proteins with binding activity, including but not limitedto antibodies and antibody derivatives. The minicell is then used togenerate a matrix and loaded in a column or tube. The solution to beseparated is mixed or passed through the column allowing the minicell tobind its target. The minicells are then separated away with the attachedsubstance.

[0689] XII.C.3. Mutagenesis

[0690] Another non-limiting example of a research application forminicells involves site directed mutagenesis studies of target proteins.In this application minicells are generated to express target proteinswith various mutations and deletions to study if function iscompromised, enhanced or has an altered specificity for ligand binding.

[0691] XII.C.4. Metabolic pathways

[0692] Another non-limiting example of research applications forminicells involves the study of metabolic rates of proteins andmetabolites. The minicell can be generated to express metabolic pathwaysand the kinetics and function of that pathway can be studied.

[0693] XII.C.5. Cell Free Production of Proteins

[0694] Another non-limiting example of a research application forminicells involves uses in cell free production of functional proteins(Jermutus et al., Recent advances in producing and selecting functionalproteins by using cell-free translation, Current Opinion inBiotechnology 9:534-548, 1999). Minicells can be prepared as a reagentused to prepare compositions for in vitro translation. As is describedin detail elsewhere herein, the composition of minicells can bemanipulated so as to be enriched for particular proteins or nulceicacids, including those involved in protein translation and foldingand/or modification of the proteins so produced into functional forms,i.e., forms having the activity of the corresponding protein as it isisolated from natural sources. Non-limiting examples of such proteinsand nulceic acids are ribosomal RNAs, ribosomal proteins, tRNAs, and thelike.

[0695] XII.C.6. Assays

[0696] Minicells could also be used in manual, semi-automated, automatedand/or robotic assays for the in vitro determinations of the compoundsof interest including, by way of non-limiting example, ligands,proteins, small molecules, bioactive lipids, drugs, heavy metals, andthe like in environmental samples (e.g., air, water, soil), blood, urineor tissue of humans or samples from non-human organisms (e.g., plants,animals, protists) for the purpose of quantifying one or more compoundsin a sample. A non-limiting example of this type of ressearchapplications is the expression on the surfaces of the minicells of areceptor such as the receptor that binds a toxin produced by Baccillusanthracis. The protein, protective antigen (PA), is a 82.7 kDa proteinthat binds one of the secreted anthrax toxins, lethal factor (LF) (seePrice, B. et al., Infection and Immunity 69: 4509-4515. 2001). Minicellsexpressing the PA protein could be used to detect LF in an environmentalsample or in human blood, urine or tissue for the purposes ofdetermining the presence of anthax. As a non-limiting example, acompetitive binding assay or an antibody-based assay could be used toindicate binding of LF in the environmental or tissue sample. Anothernon-limiting example is the use of PA-expressing minicells in a lateralflow diagnostic where interaction between the minicells and theLF-containing sample is indicated by the presence of a colored reactionproduct on a test strip.

XIII. Minicell-Based Delivery of Biologically Active Agents

[0697] XIII.A. General Considerations

[0698] The minicells of the invention are capable of encapsulatingand/or loading into a membrane a variety of substances, including butnot limited to biologically active agents, including but not limited todiagnostic and therapeutic agents. Biologically active agents include,but are not limited to, nucleic acids, e.g., DNA, RNA, gene therapyconstructs, ribozymes, antisense and other synthetic oligonucleotidesincluding those with chemical modifications; peptide nucleic acids(PNAs); proteins; synthetic oligopeptides; peptomimetics; smallmolecules; radioisotopes; antibiotics; antibodies and antibodyderivatives; and combinations and/or prodrugs of any of the preceding.

[0699] The surface of a minicell may be chemically altered in order tohave certain properties that are desirable for their use as drugdelivery agents. By way of non-limiting example, minicells may bechemically conjugated to polyethylene glycol (PEG), which provides for“stealth” minicells that are not taken as well and/or as quickly by thereticuloendothelial system (RES). Other compounds that may be attachedto minicells include without limitation polysaccharides,polynucleotides, lipopolysaccharides, lipoproteins, glycosylatedproteins, synthetic chemical compounds, and/or combinations of any ofthe preceding.

[0700] A minicell that is used to deliver therapeutic agents maycomprise and display a binding moiety. By way of non-limiting example,binding moieties used for particular purposes may be a binding moietydirected to a compound or moiety displayed by a specific cell type orcells found predominantly in one type of tissue, which may be used,among other things, to target minicells and their contents to specificcell types or tissues. A preferred binding moiety is an antibody orantibody derivative, which are described in deatil elsewhere herein.Other binding moieties include, but are not limited to, receptors,enzymes, ligands, binding peptides, fusion proteins, small moleculesconjugated to transmembrane proteins, ligands conjugated totransmembrane proteins, viral fusion proteins, and fusion/chimericproteins.

[0701] XIII.B. Cellular Uptake

[0702] In addition to binding moieties, proteins and other compoundsthat induce or enhance the uptake or fusion of the minicell with thetarget gene can be displayed on the surface of a minicell forapplications involving the delivery of therapeutic agents, gene therapy,and/or transfection or other research applications. See, generally,Adhesion Protein Protocols, Vol. 96, Dejana, E. and Corada, M., eds.,Humana Press, 1999.

[0703] XIII.B. 1. Cellular Uptake Sequences from Eukaryotic Cells

[0704] Eukaryotic adhesion receptors, which mediate intercellularadhesion, can be used as agents or targets for cellular uptake. Thereare at least three distinct classes of adhesive molecules thatleukocytes employ during their adhesive interactions (a) integrins,including but not limited to LEC-CAMS/Selectins (ELAM-1, LAM-1/Leu8/TQ1,and GMP140/PADGEM); (b) those belonging to the immunoglobulinsuperfamily including but not limited to CD2(LFA-2), CD3/TCR, CD4, CD8,CD28, CD44, CD54 (ICAM-1), ICAM-2, CD58 (LFA-3), VCAM-1, B7; and (c)Class I and II Major Histocompatability Antigens (MHC).

[0705] The adhesion receptors that belong to the integrin family andcontrol intercellular interactions are of partciular interest. At leastten different structurally related cell surface heterodimeric (alpha andbeta complexes) molecules have been defined as integrins and furtherclassified into subfamilies (Springer T. A., 1990, Nature 346:425-434;Hynes, R. O., 1987, Cell 48:549-554; Moller, G. Editor, 1990, Immunol.Rev. 114.:1-217). Each subfamily has a unique beta subunit, designatedintegrin beta1 (CD29), integrin beta2 (CD 18), and integrin beta3(CD61), each of which can associate with multiple alpha subunits, eachwith at least one di-valent cation binding site. The integrin familyincludes receptors for extracellular matrix components such asfibronectin, laminin, vitronectin, and collagen which recognizeArg-Gly-Asp in their ligands and utilize the betal or beta3 subunits(Springer T. A., 1990, Nature 346:425-434; Hynes, R. O., 1987, Cell48:549-554; Hemler, M. E., 1988, Immunol. Today 9:109-113; Patarroyo,M., and Makgoba, M. W., 1989, Scand. J. Immunol. 30:129-164; Moller, G.Editor, 1990, Immunol. Rev. 11.4:1-217). There are at least six distinctalpha subunits alph1 (CD49a), alpha2 (CD49b), alpha3 (CD49c), alpha4(CD49d), alpha5 (CD49e), and alpha6 (CD49f) capable of associating withbetal (CD29). The betal integrins are expressed on many nonhematopoieticand leukocyte cell types and are thought to play an active role intissue organization by binding to extracellular matrix components foundin many tissues and in the basement membranes underlying muscles,nervous system, epithelium and endothelium. While the expression of manybetal integrins on leukocytes requires consistent activation, theirexpression on nonhematopoietic cells does not (Hemler, M. E., 1988,Immunol. Today 9:109-113; Patarroyo, M., and Makgoba, M. W., 1989,Scand. J. Immnunol. 30:129-164). The complexity of the integrin familyhas been increased by the discovery of novel beta subunits beta3 (CD61),beta4 and beta5 that can associate with alpha 4, alpha 6, and alpha Vsubunits (Springer T. A., 1990, Nature 346:425-434; Hemler, M. E., 1988,Immunol. Today 9:109-113). This combinatorial use of alpha and betasubunits confers considerable diversity in ligand recognition and alsohelps regulate communications between the inside and outside of thecell.

[0706] By way of non-limiting example, a minicell display an adhesionreceptor, or a fusion protein that has a transmembrane domain linked toa functional portion of an adhesion receptor. Such minicells will bindto cells displaying the ligand for the adhesion receptor.

[0707] XIII.B.2. Cellular Uptake Sequences from Prokaryotes

[0708] Bacterial adhesion proteins are another source of polypetidesthat are used to stimulate uptake of minicells. See, generally, Handbookof Bacterial Adhesion: Principles, Methods, and Applications, Yuehuei H.An; Richard J. Friedman, eds., Humana Press, 2000; and Hultgren et al.,“Bacterial Adhesions and Their Assembly,” Chapter 150 in: Eschericiacoli and Salmonella typhimurium: Cellular and Molecular Biology, 2^(nd)Ed., Neidhardt, Frederick C., Editor in Chief, American Society forMicrobiology, Washington, D.C., 1996, Volume 2, pages 1903-1999, andreferences cited therein.

[0709] By way of non-limiting example, a minicell may express a proteinsuch as invasin to induce receptor mediated endocytosis (Pepe et al.,Yersinia enterocolitica invasin: A primary role in the initiation ofinfection, Proc. Natl. Acad. Sci. U.S.A. 90:6473-6477, 1993; Alrutz etal., Involvement of focal adhesion kinase in invasin-mediated uptake,Proc. Natl. Acad. Sci. U.S.A. 95:13658-13663, 1998). Invasin interactswith the Beta2 Integrin protein and causes it to dimerize. Upondimerization the Beta2 Integrin signals for an endocytotic event. Thus aminicell expressing the invasin protein will be taken up by cellsexpressing Beta2 Integrin via endocytosis.

[0710] As another non-limiting example, the pneumococcal adhesin proteinCpbA interacts with the human polyimmunoglobulin receptor (hpIgR) aseither a part of the outer surface of a bacterial cell or as a freemolecule Zhang et al. (Cell 102:827-837, 2000). The regions ofCpbA:hpIgR interaction were mapped using a series of large peptidefragments derived from CpbA. CpbA (Swiss-Prot Accession No. 030874)contains a choline binding domain containing residues 454-663 and twoN-terminal repetitive regions called R1 and R2 that are contained inresidues 97-203 and 259-365, respectively. Polypeptides containing R1and R2 interact with hpIgR, whereas polypeptides containing othersequences from CpbA do not bind to hpIgR. The R1 and/or R2 sequences ofthe CpbA polypeptide, and/or essentially identical, substantiallyidentical, or homologous amino acid sequences, are used to facilitatethe uptake of minicells by cells.

[0711] Another non-limiting example of gene delivery or transfectionusing the minicell involves the use of the type III secretion apparatusof bacteria. The type III secretion apparatus is expressed in theminicell and used to transfer genetic material to a target cell.

[0712] Other non-limiting examples of a minicell gene delivery andtransfection targeting moiety are ETA (detoxified exotoxin a) proteindelivery element described in U.S. Pat. No. 6,086,900 to Draper;Interalin and related proteins from Listeria species (Galan, AlternativeStrategies for Becoming an Insider: Lessons from the Bacterial World,Cell 103:363-366,2000); Intimin from pathogenic E. coli strains (Frankelet al., Intimin and the host cell—is it bound to end in Tir(s)? Trendsin Microbiology 9:214-218); and SpeB, streptococcal pyrogenic exotoxin B(Stockbauer et al., A natural variant of the cysteine protease virulencefactor of group A Streptococcus with an arginine-glycine-aspartic acid(RGD) motif preferentially binds human integrins α_(v)β₃ and α_(IIb)β₃Proc. Natl. Acad. Sci. U.S.A. 96:242-247, 1999).

[0713] XIII.B.3. Cellular Uptake Sequences From Viruses

[0714] Cellular uptake sequences derived from viruses include, but arenot limited to, the VP22 protein delivery element derived from herpessimplex virus-1 and vectors containing sequences encoding the VP22protein delivery element are commercially available from Invitrogen(Carlsbad, Calif.; see also U.S. Pat. No. 6,017,735 to Ohare et al.);and the Tat protein delivery element derived from the amino acidsequence of the Tat protein of human immunodeficiency virus (HIV). SeeU.S. Pat. Nos. 5,804,604; 5,747,641; and 5,674,980.

[0715] XIII.B.4. Lipids

[0716] Another non-limiting example of gene delivery and transfectionusing minicells involves minicells that have been engineered to containanionic lipids or cationic lipids (Axel et al., Toxicity, UptakeKinetics and Efficacy of New Transfection Reagents: Increase ofOligonucleotide Uptake, Jour. of Vasc. Res. 040:1-14, 2000). Many typesof lipids have been shown to induce or enhance transfection and genedelivery in a variety of cell types. Minicells containing such lipidscould be used to transfer genetic material to specific cell types.Minicells can also be engineered to express targeting proteins thatwould allow the minicell to associate tightly with a target cell, whichwill facilitate the lipid interactions and gene transfer.

[0717] Another non-limiting example of gene delivery or transfectionusing minicells involves the use of ligands to induce receptor mediatedendocytosis. By way of non-limiting example, the ligand is expressed onthe surface of the minicell, or is attached to the surface of theminicell. A minicell containing genetic material is then able toassociate with a target cell expressing the target receptor for theligand. The receptor/ligand interaction will result in the endocytosisof the minicell into the target cell where the minicell would releaseand deliver the genetic material.

[0718] Another non-limiting example of gene delivery or transfectionusing minicells involves the use of fusion proteins, such as but notlimited to viral capsid proteins. In this example the fusion proteinwould be expressed or attached to the outside of the minicell. Thefusion protein would then induce fusion of a target cell with theminicell upon contact. The contact could be initiated via randomnon-targeting events or via the use of specific targeting proteins. Inboth cases the end result would be the fusion of the minicell with atarget cell and the delivery of the genetic material.

[0719] XIII.C. Post-Targeting Expression of Cellular Uptake Sequences

[0720] Another non-limiting example of the minicell gene delivery andtransfection system using invasin involves the expression of invasinfollowing a targeting event. In this example, a minicell expresses atargeting protein that is capable of bringing the minicell in contactwith a specific target cell. Upon contact with the target cell, theminicell will be induced to transcribe and translate invasin. Theinduction is accomplished via signaling events or with a transcriptionfactor dimerization event. The minicells can be engineered to containtargeting proteins that induce protein expression only upon contact witha specific target cell. By way of non-limiting example, the invasin isexpressed only at the target cell where it induces endocytosis, thuspreventing the minicell from entering any cell but the target cell.

[0721] Proteins can be induced and expressed post contact with targetcells include but are not limited to antibodies and antibodyderivatives, receptors, enzymes, ligands, binding peptides, fusionproteins, small molecules conjugated to transmembrane proteins, ligandsconjugated to transmembrane proteins, viral fusion proteins,antibiotics, apoptotic proteins, hormones, toxins, poisons, andfusion/chimeric proteins.

[0722] XIII.D. Intracellular Targeting and Organellar Delivery

[0723] After delivery to and entry into a targeted cell, a minicell maybe designed so as to be degraded, thereby releasing the therapeuticagent it encapsulates into the cytoplasm of the cell. The minicelland/or therapeutic agent may include one or more organellar deliveryelements, which targets a protein into or out of a specific organelle ororganelles. For example, the ricin A chain can be included in a fusionprotein to mediate its delivery from the endosome into the cytosol.Additionally or alternatively, delivery elements for other organelles orsubcellular spaces such as the nucleus, nucleolus, mitochondria, theGolgi apparatus, the endoplasmic reticulum (ER), the cytoplasm, etc. areincluded Mammalian expression constructs that incorporate organellardelivery elements are commercially available from Invitrogen (Carlsbad,Calif.: pShooter™ vectors). An H/KDEL (i.e., His/Lys-Asp-Glu-Leusequence) may be incorporated into a fusion protein of the invention,preferably at the carboxy-terminus, in order to direct a fusion proteinto the ER (see Andres et al., J. Biol. Chem. 266:14277-142782, 1991; andPelham, Trends Bio. Sci. 15:483-486, 1990).

[0724] Another type of organellar delivery element is one which directsthe fusion protein to the cell membrane and which may include amembrane-anchoring element. Depending on the nature of the anchoringelement, it can be cleaved on the internal or external leaflet of themembrane, thereby delivering the fusion protein to the intracellular orextracellular compartment, respectively. For example, it has beendemonstrated that mammalian proteins can be linked to i) myristic acidby an amide-linkage to an N-terminal glycine residue, to ii) a fattyacid or diacylglycerol through an amide-or thioether-linkage of anN-terminal cysteine, respectively, or covalently to iii) aphophotidylinositol (PI) molecule through a C-terminal amino acid of aprotein (for review, see Low, Biochem. J. 244: 1-13, 1987). In thelatter case, the PI molecule is linked to the C-terminus of the proteinthrough an intervening glycan structure, and the PI then embeds itselfinto the phopholipid bilayer; hence the term “GPI” anchor. Specificexamples of proteins know to have GPI anchors and their C-terminal aminoacid sequences have been reported (see Table 1 and FIG. 4 in Low,Biochemica et Biophysica Acta, 988:427-454, 1989; and Table 3 inFerguson, Ann. Rev. Biochem., 57:285-320, 1988). Incorporation of GPIanchors and other membrane-targeting elements into the amino- orcarboxy-terminus of a fusion protein can direct the chimeric molecule tothe cell surface.

[0725] XIII.E. Minicell-Based Gene Therapy

[0726] The delivery of nucleic acids to treat diseases or disorders isknown as gene therapy (Kay et al., Gene Therapy, Proc. Natl. Acad. Sci.USA 94:12744-12746, 1997). It has been proposed to use gene therapy totreat genetic disorders as well as pathogenic diseases. For reviews, seeDesnick et al., Gene Therapy for Genetic Diseases, Acta Paediatr. Jpn.40:191-203, 1998; and Bunnell et al., Gene Therapy for InfectiousDiseases, Clinical Microbiology Reviews 11:42-56, 1998).

[0727] Gene delivery systems use vectors that contain or are attached totherapeutic nucleic acids. These vectors facilitate the uptake of thenucleic acid into the cell and may additionally help direct the nucleicacid to a preferred site of action, e.g., the nucleus or cytoplasm (Wuet al., “Delivery Systems for Gene Therapy,” Biotherapy 3:87-95, 1991).Different gene delivery vectors vary with regards to various properties,and different properties are desirable depending on the intended use ofsuch vectors. However, certain properties (for example, safety, ease ofpreparation, etc.) are generally desirable in most circumstances.

[0728] The minicells of the invention may be used as delivery agents forany therapeutic or diagnostic agent, including without limitation genetherapy constructs. Minicells that are used as delivery agents for genetherepay constructs may, but need not be, targeted to specific cells,tissues, organs or systems of an organism, of a pathogen thereof, usingbinding moieties as described in detail elsewhere herein.

[0729] In order to enhance the effectiveness of gene delivery vectorsin, by way of non-limiting example, gene therapy and transfection, it isdesirable in some applications of the invention to target specific cellsor tissues of interest (targeted cells or tissues, respectively). Thisincreases the effective dose (the amount of therapeutic nucleic acidpresent in the targeted cells or tissues) and minimizes side effects dueto distribution of the therapeutic nucleic acid to other cells. Forreviews, see Peng et al., “Viral Vector Targeting,” Curr. Opin.Biotechnol. 10:454-457, 1999; Gunzburg et al., “Retroviral VectorTargeting for Gene Therapy,” Cytokines Mol. Ther. 2:177-184, 1996.;Wickham, “Targeting Adenovirus,” Gene Ther. 7:110-114, 2000; Dachs etal., “Targeting Gene Therapy to Cancer: A Review,” Oncol. Res.9:313-325, 1997; Curiel, “Strategies to Adapt Adenoviral Vectors forTargeted Delivery,” Ann NY Acad. Sci. 886:158-171, 1999; Findeis et al.,“Targeted Delivery of DNA for Gene Therapy via Receptors,” TrendsBiotechnol. 11:202-205, 1993.

[0730] Some targeting strategies make use of cellular receptors andtheir natural ligands in whole or in part. See, for example, Cristianoet al., “Strategies to Accomplish Gene Delivery Via theReceptor-Mediated Endocytosis Pathway,” Cancer Gene Ther., Vol. 3, No.1, pp. 49-57, January-February 1996.; S. C. Philips, “Receptor-MediatedDNA Delivery Approaches to Human Gene Therapy,” Biologicals, Vol. 23,No. 1, pp. 13-6, March 1995; Michael et al., “Strategies to AchieveTargeted Gene Delivery Via the Receptor-Mediated Endocytosis Pathway,”Gene Ther., Vol. 1, No. 4, pp. 223-32, July 1994; Lin et al.,“Antiangiogenic Gene Therapy Targeting The Endothelium-Specific ReceptorTyrosine Kinase Tie2,” Proc. Natl. Acad. Sci., USA, Vol. 95, pp.8829-8834, 1998. Sudimack et al., “Targeted Drug Delivery Via the FolateReceptor,” Adv. Drug Deliv., pp. 147-62, March 2000; Fan et al.,“Therapeutic Application of Anti-Growth Factor Receptor Antibodies,”Curr. Opin. Oncol., Vol. 10, No. 1, pp. 67-73, January 1998; Wadhwa etal., “Receptor Mediated Glycotargeting,” J. Drug Target, Vol. 3, No. 2,pp. 111-27, 1995; Perales et al., “An Evaluation of Receptor-MediatedGene Transfer Using Synthetic DNA-Ligand Complexes,” Eur. J. Biochem,Vol. 1, No 2, pp. 226, 255-66, December 1994; Smith et al.,“Hepatocyte-Directed Gene Delivery by Receptor-Mediated Endocytosis,”Semin Liver Dis., Vol. 19, No. 1, pp. 83-92, 1999.

[0731] Antibodies, particularly single-chain antibodies, to surfaceantigens specific for a particular cell type may also be used astargeting elements. See, for example, Kuroki et al., “Specific TargetingStrategies of Cancer Gene Therapy Using a Single-Chain Variable Fragment(scFv) with a High Affinity for CEA,” Anticancer Res., pp. 4067-71,2000; U.S. Pat. No. 6,146,885, to Dornburg, entitled “Cell-Type SpecificGene Transfer Using Retroviral Vectors Containing Antibody-EnvelopeFusion Proteins”; Jiang et al., “In Vivo Cell Type-Specific GeneDelivery With Retroviral Vectors That Display Single Chain Antibodies,”Gene Ther. 1999, 6:1982-7; Engelstadter et al., “Targeting Human T CellsBy Retroviral Vectors Displaying Antibody Domains Selected From A PhageDisplay Library,” IIum. Gene Ther. 2000, 11:293-303; Jiang et al.,“Cell-Type-Specific Gene Transfer Into Human Cells With RetroviralVectors That Display Single-Chain Antibodies,” J. Virol1998,72:10148-56; Chu et al., “Toward Highly EfficientCell-Type-Specific Gene Transfer With Retroviral Vectors DisplayingSingle-Chain Antibodies,” J. Virol 1997, 71:720-5; Chu et al.,“Retroviral Vector Particles Displaying The Antigen-Binding Site Of AnAntibody Enable Cell-Type-Specific Gene Transfer,” J. Virol 1995,69:2659-63; and Chu et al., “Cell Targeting With Retroviral VectorParticles Containing Antibody-Envelope Fusion Proteins,” Gene Ther.1994, 1:292-9.

[0732] Minicells are used to deliver DNA-based gene therapy to targetedcells and tissues. Double minicell transformants are used not only totarget a particular cell/tissue type (e.g. HIV-infected T-cells) but arealso engineered to fuse with and enter targeted cells and deliver aprotein-based toxin (e.g., antibiotic, or pro-apoptotic gene like Bax),an antisense expression construct (e.g., antisense to a transcriptionfactor), or antisense oligonucleotides (e.g., antisense to ananti-apoptotic gene such as Bcl-2. The doubly-transformed minicellsexpress not only these cell death promoters, but also only targetparticular cells/tissues, thus minimizing toxicity and lack ofspecificity of gene therapy vectors. By “doubly-transformed” it is meantthat the minicell comprises 2 expression elements, one eubacterial, theother eukaryotic. Alternaively, shuttle vectors, which compriseeubacterial and eukaryotic expression elements in one vector, may beused.

[0733] Minicell-based gene therapy is used to deliver expressionplasmids that could correct protein expression deficiencies orabnormalities. As a non-limiting example, minicell inhalants aretargeted to pulmonary alveolar cells and are used to deliver chloridetransporters that are deficient or otherwise material in cysticfibrosis. Protein hormone deficiencies (e.g., dwarfism) are corrected byminicell expression systems (e.g., growth hormone expression inpituitary cells). Duchene's muscular dystrophy is characterized by amutation in the dystrophin gene; this condition is corrected byminicell-based gene therapy. Angiogenesis treatment for heart patientsis made effective by FGF or VGEF-producing minicells targeted to theheart. In this case, plasmid-driven over-expression of these grownfactors is preferred.

XIV. Therapeutic Uses of Minicells

[0734] In addition to minicell-based gene therapy, minicells can be usedin a variety of therapeutic modalities. Non-limiting examples of thesemodalities include the following applications.

[0735] XIV.A. Diseases and Disorders

[0736] Diseases and disorders to which the invention can be appliedinclude, by way of non-limiting example, the following.

[0737] Diseases and disorders that involve the respiratory system, suchas cystic fibrosis, lung cancer and tumors, asthma, pathogenicinfections, allergy-related diseases and disorders, , such as asthma;allergic bronchopulmonary aspergillosis; hypersensitivity pneumonia,eosinophilic pneumonia; emphysema; bronchitis; allergic bronchitisbronchiectasis; cystic fibrosis; hypersensitivity pneumotitis;occupational asthma; sarcoid, reactive airway disease syndrome,interstitial lung disease, hyper-eosinophilic syndrome, parasitic lungdisease and lung cancer, asthma, adult respiratory distress syndrome,and the like;

[0738] Diseases and disorders of the digestive system, such as those ofthe gastrointestinal tract, including cancers, tumors, pathogenicinfections, colitis; ulcerative colitis, diverticulitis, Crohn'sdisease, gastroenteritis, inflammatory bowel disease, bowel surgeryulceration of the duodenum, a mucosal villous disease including but notlimited to coeliac disease, past infective villous atrophy and short gutsyndromes, pancreatitis, disorders relating to gastroinstestinalhormones, Crohn's disease, and the like;

[0739] Diseases and disorders of the skeletal system, such as spinalmuscular atrophy, rheumatoid arthritis, osteoarthritis, osteoporosis,multiple myeloma-related bone disorder, cortical-striatal-spinaldegeneration, and the like;

[0740] Autoimmune diseases, such as Rheumatoid arthritis (RA), multiplesclerosis (MS), Sjogren's syndrome, sarcoidosis, insulin dependentdiabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis,ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis,psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease andulcerative colitis amyotrophic lateral sclerosis, multiple sclerosis,autoimmune gastritis, systemic lupus erythematosus, autoimmune hemolyticanemia, autoimmune neutropenia, systemic lupus erythematosus, graft vs.host disease, bone marrow engraftment, some cases of Type I diabetes,and the like;

[0741] Neurological diseases and disorders, such as depression, bipolardisorder, schizophrenia, Alzheimer's disease, Parkinson's disease,familial tremors, Gilles de la Tourette syndrome, eating disorders,Lewy-body dementia, chronic pain and the like;

[0742] Pathological diseases and resultant disorders such as bacterialinfections such as infection by Escherichia, Shigella, Salmonella;sepsis, septic shock, and bacteremia; infections by a virus such as HIV,adenovirus, smallpox virus, hepatovirus, and the like; and AIDS-relatedencephalitis, HIV-related encephalitis, chronic active hepatitis, andthe like;

[0743] Proliferative disease and disorders, such as acute lymphoblasticleukemia, acute myelogenous leukemia, chronic myelogenous leukemia,metastatic melanoma, Kaposi's sarcoma, multiple myeloma, breast cancer,anal cancer, vulvar cancer, and the like; and

[0744] Various diseases, disorders and traumas including, but notlimited to, apoptosis mediated diseases, inflammation, cerebralischemia, myocardial ischemia, aging, sarcoidosis, granulomatouscolitis, scleroderma, degenerative diseases, necrotic diseases,alopecia, neurological damage due to stroke, diffuse cerebral corticalatrophy, Pick disease, mesolimbocortical dementia, thalamicdegeneration, Huntington chorea, cortical-basal ganglionic degeneration,cerebrocerebellar degeneration, familial dementia with spasticparaparesis, polyglucosan body disease, Shy-Drager syndrome,olivopontocerebellar atrophy, progressive supranuclear palsy, dystoniamusculorum deformans, Hallervorden-Spatz disease, Meige syndrome,acanthocytic chorea, Friedreich ataxia, Holmes familial corticalcerebellar atrophy, Gerstmann-Straussler-Scheinker disease, progressivespinal muscular atrophy, progressive balbar palsy, primary lateralsclerosis, hereditary muscular atrophy, spastic paraplegia,glomeralonephritis, chronic thyroiditis, Grave's disease,thrombocytopenia, myasthenia gravis, psoriasis, peroneal muscularatrophy, hypertrophic interstitial polyneuropathy, heredopathia atacticapolyneuritiformis, optic neuropathy, and ophthalmoplegia.

[0745] A variety of diseases and disorders caused or exacerbated bypathogens may be treated using the invention. For a comprehensivedescription of pathogens and associated diseases and disorders, seeZinsser Microbiology, 20th Ed., Joklik, ed., Appelton-Century-Crofts,Norwalk, Conn., 1992, and references cited therein.

[0746] Minicells could also be used for replacement therapy (via genetherapy) in a variety of conditions known to be caused by protein orproteins that are either absent (e.g. Duchene's Muscular Dystrophy),reduced in level (Dwarfism) or abberant (Sickle-cell anemia).

[0747] For a comprehensive description of diseases and disorders thatmay be treated using the invention, see The Merck Manual of Diagnosisand Therapy, 17th Ed., Beers et al., eds.; published edition, Merck andCo., Rahway, N.J., 1999; on-line edition, Medical Services, Usmedsa,USHH, http://www.merck.comlpubs/mmanual/, and references cited therein.

[0748] XIV.B. Removal of Toxins and Pathogens by Selective Absorption

[0749] When introduced into the bloodstream of an animal,receptor-displaying minicells bind and absorb toxic compounds, therebyremoving such compounds from the general circulation. A therapeuticbenefit ensues as the bound toxic compound cannot access the cells uponwhich it would otherwise exert its toxic effect.

[0750] Minicells expressing receptors for toxic substances areintroduced IV in order to remove those toxins from the blood. Onenon-limiting example is in the treatment of sepsis. In one embodiment, afusion protein is formed from the transmembrane domain of the EGFreceptor or toxR and a known soluble receptor for LPS(lipopolysaccharide), such as the LBP (lipopolysaccharide bindingprotein) or the extracellular domain of CD14 receptor protein, both ofwhich bind the LPS bacterial endotoxin. These minicells inactivate LPSby initially binding to it and preventing LPS binding to naturallyoccurring CD14 receptors on heart cells and other cells involved in theendotoxic shock response. Eventually, the minicell-LPS complex iscleared from the blood by macrophages and other components of the immunesystem.

[0751] In another embodiment, minicells expressing receptors for toxicdrugs (e.g., morphine) are used to treat drug overdoses. In otherembodiments, minicells of the invention are used to express receptors tovenoms (e.g., snake venom) or poisons (e.g., muscarinic receptorexpression for the treatment of muscarine poisoning). In otherembodiments, minicells of the invention expressing EDGRs are used toclear the blood of toxins and other undesirable compounds.

[0752] As another non-limiting example, minicells that bind pathogensare used to treat disease. Minicells, and pathogens bound thereto, maybe ingested by human neutrophils and thus serve as an adjuvant totherapeutic processes mediated by neutrophils (Fox et al., Fate of theDNA in plasmid-containing Escherichia coli minicells ingested by humanneutrophils, Blood 69:1394-400, 1987). In a related modality, minicellsare used to bind compounds required for the growth of a pathogen.

[0753] XIV.C. Antiviral Therapy

[0754] In one modality, minicells of the invention are used as “sponges”for the selective absorption of any viral particle in the body. Withoutbeing limited to the following examples, minicells expression receptorsor antibodies selectively directed against viruses such as HIV,Hepatitis B and smallpox are used.

[0755] For the treatment of viremia, viruses are cleared from the bloodby absorption during dialysis or by IV injection of minicells expressingtargets for viral receptors. As the minicells interact with blood-boruevirus particles, there is an initial reduction of host cell infection byvirtue of the minicell-viral complexes that are formed. Since viralparticles attach to and/or enter the minicell, they are not activebecause of the lack of machinery needed for viral replication in theminicells. The virus infected minicells are then cleared from the systemby macrophages and processed by the immune system.

[0756] Certain retroviruses that infect particular host cells expressviral proteins on the surfaces of the infected cells. HIV infection ofT-cells is one non-limiting example of this. The viral protein, GP120,is expressed on the surfaces of infected T-cells (Turner et al.,Structural Biology of HIV, J. Mol. Biol. 285:1-32, 1999). Minicellsexpressing CD4 act as anti-GP120 minicells not only to target virusparticles in an infected patient, but also to identify infected T-cells.It may be desirable to also express co-receptors such as CCR5, CXR4 orARD (Dragic, An overview of the determinants of CCR5 and CXCR4co-receptor function, J. Gen. Virol. 82:1807-1814, 2001). The minicellsare then used to kill the infected T-cells, or to inhibit viralreplication and/or virion assembly.

[0757] In another non-limiting example of anti-pathogen therapy,minicells can by used to express bacterial surface antigens on theirsurfaces that facilitate cellular uptake of the minicell byintracellular pathogens such as Mycobacterium tuberculosis (thecausative agent of tuberculosis), Rickettsiae, or viruses. In this“smart sponge” approach, selective absorption is accompanied byinternalization of the pathogen by minicells. Destruction of thepathogen follows as a result of a combination of intraminicell digestionof pathogens and/or by the eventual processing of the virus-containingminicell by the cellular immune system of the patient.

[0758] XIV.D. Antibacterial and Antiparasitic Applications

[0759] Minicells may be used to kill pathogenic bacteria, protozoans,yeast and other fungi, parasitic worms, viruses and other pathogens bymechanisms that either do or do not rely on selective absorption.Antibiotics can be delivered to pathogenic organisms after first beingtargeted by the proteins or small molecules on the surfaces of theminicells that promote binding of the minicells to the surfaces of thepathogen. Fusion or injection of minicell contents into the pathogeniccell can result in the death or disablement of the pathogen and thuslower the effective dose of an antibiotic or gene therapeutic agent.Delivery of antibiotics tethered to or encapsulated by the minicellswill reduce the effective dose of an antibiotic and will reduce itselimination by the renal system. In the case of delivering encapsulatedmolecules (e.g., antibiotics), purified/isolated minicells expressingmembrane-bound proteins for targeting can be incubated with themolecules in vitro prior to administration. This would be particularlyapplicable to the use of protoplast minicells or poroplast minicellsthat have their outer membrane and cell wall or outer membrane onlyremoved, respectively, thus facilitating the diffusion of the smallmolecule into the intact minicell.

[0760] Without being limited by the following example, minicells can beuse as antibacterial agents by expressing on the surfaces of theminicells antigens, receptors, antibodies, or other targeting elementsthat will target the minicell to the pathogenic organism and facilitatethe entry of plasmids, proteins, small molecules in order to gain accessto or entry into the organism. Antibiotics may be encapsulated byminicells post isolation from the parent strain so that the antibioticwill not be effective against the minicell-producing bacteria itself.Since minicells are not able to reproduce, the antibiotic will not belethal to the minicell delivery vehicle, but only to the targetedpathogen. In another non-limiting example, lyosgenic factors e.g.,complement may be expressed on the surfaces of the minicells orencapsulated by same as to promote lysis of the pathogen.

[0761] Minicells can also be engineered to express toxic proteins orother elements upon binding to the pathogen. Induction of minicellprotein expression can be an event that is coincident with targeting ortriggered by minicell binding to the target pathogen, thus makingminicells toxic only when contact is made with the pathogenic organism.Minicells can be engineered to express fusion/chimeric proteins that aretethered to the membrane by transmembrane domains that have signalingmoieties on the cytoplasmic surfaces of the minicells, such as kinasesor transcription factors. In one non-limiting example, a minicell fusionmembrane-bound protein could be expressed containing an extracellulardomain with a receptor, scFv, or other targeting protein that binds tothe pathogen. The second segment of the chimera could be a transmembranedomain of a protein such as the EGF receptor or ToxR (that would tetherthe fusion protein to the membrane). Importantly, the cytoplasmic domainof the fusion protein could be a kinase that phosphorylates a bacterialtranscription factor present in the minicell or could be fused to atranscription factor that would be expressed on the cytoplasmic surfaceof the minicell. The expression plasmid that was previously introducedinto the minicells would then be activated by promoters utilizing theactivated bacterial transcription factor pre-existing in the minicellsor that which may be introduced by the minicell. Without being limitedto the following example, the binding event could be signaled by afusion protein containing elements of a receptor (e.g., EGF) or by anadhesion protein (e.g., an integrin) that require oligomerization. Inthe example of the use of integrins, bacterial or other transcriptionfactors that also require dimerization could be cloned as fusionproteins such that the binding event would be signaled by a dimerizationof two or more identical recombinant chimeric proteins that haveassociation-dependent transcription factors tagged to the C-terminus ofthe fusion protein. The minicells would only be toxic when contact ismade with the pathogen.

[0762] The proposed mechanism of induction coincident with targeting isnot limited to the antiparasitic uses of minicells but can be used inother therapeutic situations where minicells are used to expressproteins of therapeutic benefit when directed against eucaryotic cellsof the organism (e.g., kill cancer cells with protein toxins expressedonly after binding of the minicell to the cancer cell).

[0763] Transfer of DNA-containing plasmids or other expression element,antisense DNA, etc. may be used to express toxic proteins in the targetcells or otherwise inhibit transcription and/or translation in thepathogenic organism or would otherwise be toxic to the cell. Withoutbeing limited by the following example, minicells can be used totransfer plasmids expressing growth repressors, DNAses, or otherproteins or peptides (e.g., pro-apoptotic) that would be toxic to thepathogen.

[0764] XIV.E. Cancer Therapy

[0765] Fusion proteins expressed in minicells are used for cancertherapy. In a non-limiting example, phage display antibody libraries areused to clone single chain antibodies against tumor-associated(tumor-specific) antigens, such as MUCH-1 or EGFvIII. Fusion proteinsexpressing these antibodies, and further comprising a single-passtransmembrane domain of an integral membrane protein, are used to“present” the antibody to the surface of the minicells. Injectedminicells coated with anti-tumor antibodies target the tumor and deliverpro-apoptotic genes or other toxic substances to the tumor. Theminicells are engulfed by the tumor cells by processes suchreceptor-mediated endocytosis (by, e.g., macrophages). By way ofnon-limiting example, toxR-invasin could be expressed on the surfaces ofthe minicells to promote endocytosis through the interaction betweeninvasin and beta2-integrins on the surfaces of the target cells.

[0766] Fusion proteins possessing viral fusion-promoting proteinsfacilitate entry of the minicell to the tumor cell for gene therapy orfor delivery of chemotherapy bioactive proteins and nucleic acids. Inthese and similar applications, the minicell may contain separateeukaryotic and eubacterial expression elements, or the expressionelements may be combined into a single “shuttle vector.”

XV. Diagnostic Uses of Minicells

[0767] Minicells are transformed with plasmids expressing membrane-boundproteins, such as receptors, that bind to specific molecules in aparticular biological sample such as blood, urine, feces, sweat, salivaor a tissue such as liver or heart. Minicells can also be used fordelivery of therapeutic agents across the blood-brain barrier to thebrain. This modality is used, by way of non-limiting example, forimaging purposes, and for the delivery of therapeutic agents, e.g.,anti-depressants, and agents for the treatment of cancer, obesity,insomnia, schizophrenia, compulsive disorders and the like. Recombinantexpression systems are incorporated into minicells where theplasmid-driven protein expression construct could be the produce asingle gene product or a fusion protein, such as a soluble protein forthe particular ligand fused with a transmembrane domain of a differentgene. The fusion protein then acts as a membrane bound receptor for aparticular ligand or molecule in the sample. Conventional cloningtechniques (e.g., PCR) are used to identify genes for binding proteins,or phage display is used to identify a gene for a single-strandedvariable antibody gene expressing binding protein for a particularligand. The protein product is preferably a soluble protein. Byconstructing a plasmid containing this gene plus the transmembranedomain of a known single-pass membrane protein such as that of the EGFreceptor, a fusion protein may be expressed on the surfaces of theminicells as an integral membrane protein with an extracellular domainthat is preferably capable of binding ligand.

[0768] In another type of fusion protein, the transmembrane domain ofthe EGF receptor is fused to a known soluble receptor for a particularligand, such as the LBP (lipopolysaccharide binding protein) or theextracellular domain of CD14 receptor protein, both of which bind thebacterial endotoxin, LPS (lipopolysaccharide). The LBP/EGF cr CD14/EGFfusion protein is used to measure LPS in the serum of patients suspectedof sepsis.

[0769] The minicell system is used to express receptors such as those ofthe EDG (endothelial cell differentiation gene) family (e.g., EDG 1-9)that recognize sphingolipids such as sphingosine-1-phosphate (S1P),sphingosylphosphoryl choline (SPC) and the lysophospholipid,lysophosphatidic acid (LPA). Since these proteins are 7-pass integralmembrane proteins, no additional transmembrane domains of anotherprotein are needed, and the receptor protein is thus not a fusionprotein.

[0770] Truncated or mutant forms of a protein of interest are useful ina diagnostic assay. For example, a protein that is an ligand-bindingenzyme can be altered so as to bind its substrate of interest but can nolonger convert substrate into product. One example of this applicationof minicell technology is the expression of a truncated or mutant lacticdehydrogenase which is able to bind lactic acid, but is not able toconvert lactic acid to pyruvate. Similarly, hexokinase deriviatives areused in minicells for glucose monitoring.

[0771] Minicells as diagnostic tools can be used either in vitro or invivo. In the in vitro context, the minicells are used in an ELISA formator in a lateral flow diagnostic platform to detect the presence andlevel of a desired analyte. A sample (tissue, cell or body fluid sample)is taken and then tested in vitro. One advantage of the minicell systemin detecting substances in tissue, cells or in body fluids is that theminicells can be used in vitro assays where the minicell is labeled witheither a radioactive or fluorescent compound to aid in its detection ina an ELISA format or lateral flow platform. Thus, the use of secondaryantibody detection systems is obviated.

[0772] As an in vivo diagnostic, minicells can be radiolabeled. Onemethod of labeling is to incubate minicells for a short time (about 8hr) with a T_(½) tracer (e.g., Tn99M) that is useful for detecting tumormetastases. The Tn99M accumulates in cells and loads into minicellsafter isolation or into the parent bacteria during growth phase. AsTn99M is oxidized by either the parent E. coli strain or by theminicells after isolation, the Tn99M is retained by the cell.Iodine-labled proteins may also be used (Krown et al., TNF-alphareceptor expression in rat cardiac myocytes: TNF-alpha inhibition ofL-type Ca2+ transiets, FEBS Letters 376:24-30, 1995).

[0773] One non-limiting example of in vivo detection of cancer makinguse of radiolabeled minicells is the use of the minicells to expresschimeric membrane-bound single-chain antibodies against tumor-specificantigens (TSA) expressed on malignant melanoma or other transformedcells. Such TSAs include, but are not limited to, the breast cancerassociated MUC1 antigen and variant forms of the EGFR (EGFvIII). By wayof non-limiting example, minicells expressing antibodies to melanomacells can be injected (IV) into a patient and then subjected to CAT scanof the lymphatic drainage in order to determine if a metastasis hasoccurred. This diagnostic technique obviates the need for lymph nodedissection.

[0774] Another example of an in vivo diagnostic is to use the minicellsystem to express antibodies against oxidized low-density lipoproteins(LDL). Oxidized LDLs are associated with atherogenic plaques.Radiolabeled minicells (prepared as above) are injected IV into a personprior to nuclear imaging for image enhancement. MRI image contrastenhancement is performed by preparing minicells complexed (loaded) withcontrast enhancers such as paramagnetic relaxivity agents and magneticsusceptibility agents.

[0775] In diagnostic as well as other applications, minicellspreferentially detect a diagnostic marker, i.e., a marker associatedwith a disease or disorder. A diagnostic marker is statistically morelike to occur in individuals sufferening from a disease than in thosewho are not diseased. Preferably, a diagnostic marker directly causes oris produced during a disease; however, the association may be no morethan a correlation.

XVI. Drug Discovery (Screening) with Minicells

[0776] XVI.A. Assays

[0777] Minicells can be used in assays for screening pharmacologicalagents. By way of non-limiting example, the minicell system provides anenvironment for the expression of GPCRs and studies of their ligandbinding kinetics. Such GPCR's include any member the EndothelialDifferentiation Gene (EDG) receptor family. GPCRs may participate inneoplastic cell proliferation, angiogenesis and cell death. Smallmolecules that either activate or inhibit the action of these GPCRs canbe used in therapeutic interaction.

[0778] Assays are performed to determine protein expression and proteinfunction. For example, the production of the protein can be followedusing protein ³⁵S-Met labeling. This is performed by providing the cellonly methionine that is labeled with ³⁵S. The cells are treated withIPTG to induce protein expression, and the ³⁵S-Met is incorporated intothe protein. The cells are then lysed, and the resulting lysates wereelectrophoresed on an SDS gel and exposed to autoradiography film.

[0779] Another technique for assessing protein expression involves theuse of western blots. Antibodies directed to various expressed proteinsof interest have been generated and many are commercially available.Techniques for generating antibodies to proteins or polypeptides derivedtherefrom are known in the art (see, e.g., Cooper et al., Section III ofChapter 11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel etal., eds., John Wiley and Sons, New York, 1992, pages 11-22 to 11-46).Standard western blot protocols, which may be used to show proteinexpression from the expression vectors in minicells and other expressionsystems, are known in the art. (see, e.g., Winston et al., Unit 10.7 ofChapter 10 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel etal., eds., John Wiley and Sons, New York, 1992, pages 10-32 to 10-35).

[0780] The amount of functional protein produced from a minicellexpression system is determined through the use of binding studies.Ligands for the proteins of interest are used to show specific bindingin the minicell system. Radiolabeled ligand is incubated with cellsexpressing the protein, in this case, a receptor for TNF-alpha. Thecells are then centrifuged and the radioactivity counted in ascintillation counter. The amount of ligand that is bound reflects theamount of functional protein that is present in the sample.

[0781] By way of non-limiting example, the minicell system can be madeto express EDGRs for the purpose of screening combinatorial chemistrylibraries for molecules that enhance EDG activity. EDG activity isassayed in the minicell environment in several ways. One way is tocrystallize minicells expressing an EDG protein (or any membrane-boundprotein of choice) and then measure changes in the crystal structure todetect novel ligands. Circular dichroism (CD), x-ray diffraction,electron spin resonance (EPR) or other biophysical approaches are usedto probe the structure of proteins in the minicell context. Additionallyor alternately, minicells are produced that express not only the EDGR,but also express G-proteins (i.e., double transformants). An assaysystem involving GTP binding and hydrolysis is used to identify andassess which small molecules in the combinatorial chemistry libraryserve as activating ligands for EDG. The minicell expression system isused in in vitro binding assays and in high throughput drug screenings.The expression of mutant or truncated isoforms of proteins are used forfunctional analyses in order to discover inactive or overactive proteinsfor potential use in diagnostics or therapeutics.

[0782] XVI.B. High-Throughput Screening (HTS)

[0783] HTS typically uses automated assays to search through largenumbers of compounds for a desired activity. Typically HTS assays areused to find new drugs by screening for chemicals that act on aparticular enzyme or molecule. For example, if a chemical inactivates anenzyme it might prove to be effective in preventing a process in a cellthat causes a disease. High throughput methods enable researchers to tryout thousands of different chemicals against each target very quicklyusing robotic handling systems and automated analysis of results.

[0784] As used herein, “high throughput screening” or “HTS” refers tothe rapid in vitro screening of large numbers of compounds (libraries);generally tens to hundreds of thousands of compounds, using roboticscreening assays. Ultra high-throughput Screening (uHTS) generallyrefers to the high-throughput screening accelerated to greater than100,000 tests per day.

[0785] To achieve high-throughput screening, it is best to house sampleson a multicontainer carrier or platform. A multicontainer carrierfacilitates measuring reactions of a plurality of candidate compoundssimultaneously. Multi-well microplates may be used as the carrier. Suchmulti-well microplates, and methods for their use in numerous assays,are both known in the art and commercially available.

[0786] Screening assays may include controls for purposes of calibrationand confirmation of proper manipulation of the components of the assay.Blank wells that contain all of the reactants but no member of thechemical library are usually included. As another example, a knowninhibitor (or activator) of an enzyme for which modulators are sought,can be incubated with one sample of the assay, and the resultingdecrease (or increase) in the enzyme activity determined according tothe methods herein. It will be appreciated that modulators can also becombined with the enzyme activators or inhibitors to find modulatorswhich inhibit the enzyme activation or repression that is otherwisecaused by the presence of the known the enzyme modulator. Similarly,when ligands to a sphingolipid target are sought, known ligands of thetarget can be present in control/calibration assay wells.

[0787] The minicells of the invention are readily adaptable for use inhigh-throughput screening assays for screening candidate compounds toidentify those which have a desired activity, e.g., inhibiting an enzymethat catalyzes a reaction that produces an undesirable compound,inhibiting function of a receptor independent of ligand interference, orblocking the binding of a ligand to a receptor therefor. The compoundsthus identified can serve as conventional “lead compounds” or canthemselves be used as therapeutic agents.

[0788] The methods of screening of the invention comprise usingscreening assays to identify, from a library of diverse molecules, oneor more compounds having a desired activity. A “screening assay” is aselective assay designed to identify, isolate, and/or determine thestructure of, compounds within a collection that have a preselectedactivity. By “identifying” it is meant that a compound having adesirable activity is isolated, its chemical structure is determined(including without limitation determining the nucleotide and amino acidsequences of nucleic acids and polypeptides, respectively) the structureof and, additionally or alternatively, purifying compounds having thescreened activity). Biochemical and biological assays are designed totest for activity in a broad range of systems ranging fromprotein-protein interactions, enzyme catalysis, small molecule-proteinbinding, agonists and antagonists, to cellular functions. Such assaysinclude automated, semi-automated assays and HTS (high throughputscreening) assays.

[0789] In HTS methods, many discrete compounds are preferably tested inparallel by robotic, automatic or semi-automatic methods so that largenumbers of test compounds are screened for a desired activitysimultaneously or nearly simultaneously. It is possible to assay andscreen up to about 6,000 to 20,000, and even up to about 100,000 to1,000,000 different compounds a day using the integrated systems of theinvention.

[0790] Typically in HTS, target molecules are contained in each well ofa multi-well microplate; in the case of enzymes, reactants are alsopresent in the wells. Currently, the most widely established techniquesutilize 96-well microtiter plates. In this format, 96 independent testsare performed simultaneously on a single 8 cm×12 cm plastic plate thatcontains 96 reaction wells. One or more blank wells contains all of thereactants except the candidate compound. Each of the non-standard wellscontain at least one candidate compound.

[0791] These wells typically require assay volumes that range from 50 to500 ul. In addition to the plates, many instruments, materials,pipettors, robotics, plate washers and plate readers are commerciallyavailable to fit the 96-well format to a wide range of homogeneous andheterogeneous assays. Microtiter plates with more wells, such as384-well microtiter plates, are also used, as are emerging methods suchas the nanowell method described by Schullek et al. (Anal Biochem., 30246, 20-29, 1997).

[0792] In one modality, screening comprises contacting a sphingolipidtarget with a diverse library of member compounds, some of which areligands of the target, under conditions where complexes between thetarget and ligands can form, and identifying which members of thelibraries are present in such complexes. In another non limitingmodality, screening comprises contacting a target enzyme with a diverselibrary of member compounds, some of which are inhibitors (oractivators) of the target, under conditions where a product or areactant of the reaction catalyzed by the enzyme produce a detectablesignal. In the latter modality, inhibitors of target enzyme decrease thesignal from a detectable product or increase a signal from a detectablereactant (or vice-versa for activators).

[0793] Minicells of the invention expressing and/or displaying a proteinare used for screening assays designed to identify agents that modulatethe activity of the target protein. Such assays include competitiveinhibition binding assays for high throughput assays. Competitiveinhibition assays include but are not limited to assays that screenagents against a specific target protein to identify agents thatinhibit, interfere, block, or compete with protein-ligand interactions,protein-protein interactions, enzymatic activity, or function of aspecific protein. Examples of competitive inhibition include but are notlimited to the development of neutral inhibitors of the serine proteasefactor Xa that were discovered using a high throughput screening assayusing a compound library (Carr et al, Neutral inhibitors of the serineprotease factor Xa, Bioorg Med Chem Lett 11, 2001), the design andcharacterization of potent inhibitors for the human oxytocin receptor(Seyer et al, Design, synthesis and pharmacological characterization ofa potent radio iodinated and photoactivatable peptidic oxytocinantagonist, J Med Chem. 44:3022-30, 2001), and the identification ofnon-peptide somatostatin antagonists of the sst(3) protein (Thurieau etal, Identification of potent non-peptide somatostatin antagonists withsst(3) selectivity, J Med Chem. 44:2990-3000, 2001).

[0794] High throughput competitive inhibition assays are designed toidentify agents that inhibit a specific target protein. Such assaysinclude but are not limited to ones that measure enzymatic activity,protein-ligand interactions, protein-protein interactions and other,functions of proteins. Minicells that express and/or display a specificprotein could be used in all types of competitive inhibition assays.

[0795] One non-limiting example of high throughput competitiveinhibition screening using minicells for the purpose of this patentinvolves the competitive inhibition of known ligands. The ligand isattached to but not limited to a flourophore, fluorescent protein, tagssuch as 6×His tag or FLAG tag, chromophores, radiolabeled proteins andmolecules, binding moieties such as avidin and strepavidin, voltagesensitive dies and proteins, bioluminescent proteins and molecules, orfluorescent peptides. The target protein, which binds the tagged ligand,is expressed and stably displayed by the minicell. When the ligand isadded to the minicell solution the ligand binds to the target protein.Following a wash the interaction is detected via the flourophore,fluorescent protein, tag, or fluorescent peptide. The ligand-boundminicells could either be centrifuged (taking advantage of thesedimentation properties of the minicell particle) or immunoprecipitatedwith an antibody against an antigen expressed on the minicell membraneor the minicells can be adsorbed/fixed to a substrate such as a standard96 well plate. The competitive inhibition assay is carried out by addingagents to the minicell mix either before, together or after the ligandis added. Thus if the agent is a competitive inhibitor of the ligand tothe target protein the ligand will be washed away from the minicellbecause it is not associated with the target protein. The agent preventsbinding and thus eliminated the detection signal from the minicell.

[0796] Minicells of this invention are used in “functional screening HTSassays”. Functional screening assays are defined as assays that provideinformation about the function of a specific target protein. Functionalassays screen agents against specific target proteins to identify agentsthat either act as antagonist or as an agonist against the protein.Functional assays require that the target protein be in an environmentthat allows it to carry out its natural function. Such functions includebut are not limited to G-proteins coupling with a GPCR, enzymaticactivity such as phosphorlyation or proteolysis, protein-proteininteraction, and transport of molecules and ions.

[0797] Functional assays screen agents against proteins which arecapable of natural function. Target proteins used in functional studiesmust carry out a function that is measurable. Examples of proteinfunctions that are measurable include but are not limited to the use ofFluorescent Resonance Energy Transfer (FRET) to measure the G-proteincoupling to a GPCR (Ruiz-Velasco et al., Functional expression and FRETanalysis of green fluorescent proteins fused to G-protein subunits inrat sympathetic neurons, J Physiol. 537:679-692, 2001; Janetopoulos etal., Receptor-mediated activation of heterotrimeric G-proteins in livingcells, Science 291:2408-2411, 2001); Bioluminescence Resonance EnergyTransfer (BRET) to assay for functional ligand induced G-proteincoupling to a target GPCR (Menard, L. Bioluminescence Resonance EnergyTransfer (BRET): A powerful platform to study G-protein coupledreceptors (GPCR) activity in intact cells, Assay Development, Nov.28-30, 2001), the use of florescent substrates to measure the enzymaticactivity of proteases (Grant, Designing biochemical assays for proteasesusing fluorogenic substrates, Assay Development, Nov. 28-30, 2001); andthe determination of ion channel function via the use of voltagesensitive dies (Andrews et al, Correlated measurements of free and totalintracellular calcium concentration in central nervous system neurons,Microsc Res Tech. 46:370-379, 1999).

[0798] One non-limiting example of high throughput functional screeningassay using minicells for the purpose of this patent involves thefunctional coupling of GPCRs to their respective G-protein. Upon ligandbinding, voltage polarization, ion binding, light interaction and otherstimulatory events activate GPCRs and cause them to couple to theirrespective G-protein. In a minicell, both the GPCR and its respectiveG-proteins can be simultaneously expressed. Upon activation of the GPCRthe coupling event will occur in the minicell. Thus by detecting thiscoupling in the minicell, one could screen for agents that bind GPCRs toidentify antagonists and agonists. The antagonists are identified usinginhibition assays that detect the inhibition of function of the GPCR.Thus the agent interacts with the GPCR in a way that it inhibits theGPCR from being activated. The agonists are identified by screening foragents that activate the GPCR in the absence of the natural activator.

[0799] The detection of GPCR activation and coupling in a minicell isaccomplished by using systems that generate a signal upon coupling. Onenon-limiting example involves the use of BRET or FRET. These systemsrequire that two fluorescent or bioluminescent molecules or proteins bebrought into close contact. Thus by attaching one of these molecules orproteins to the GPCR and one to the G-protein, they will be broughttogether upon coupling and a signal will be generated. This signal canbe detected using specific detection equipment and the coupling eventcan be monitored. Thus the function of the GPCR can be assayed and usedin functional assays in minicells.

[0800] Another non-limiting functional assay for GPCRs and otherproteins in minicells involves the use of transcription factors. Manybacterial transcription factors and eukaryotic transcription factorsrequire dimerization for activation. By attaching one subunit of atranscription factor to a GPCR and the other subunit to a G-protein, thesubunits will dimerize upon coupling of the GPCR to the G-proteinbecause they will be brought into close contact. The dimerizedtranscription factor will then be activated and will act on its targetepisomal DNA. In the minicell system the episomal DNA target will be aplasmid that encodes for proteins that provide a signal for detection.Such proteins include but are not limited to luciferase; greenfluorescent protein (GFP), and derivatives thereof such as YFP, BFP,etc.; alcohol dehydrogenase, and other proteins that can be assayed forexpression. The activation of the GPCR will result in coupling andactivation of the transcription factor. The transcription factor willthen induce transcription and translation of specific detector proteins.Thus the activation of the GPCR will be monitored via the expression ofthe detector protein.

[0801] In another modality, the transcription factor can inhibitexpression in the minicell system and thus allowing for the screening ofconstitutively active GPCRs and proteins. For example if the GPCR wereconstitutively active then the transcription factor to use would be onethat inhibits transcription and translation. Thus agents could bescreened against the constitutively active GPCR to identify agents thatcaused the constitutively active GPCR to uncouple. The uncoupling willresult in the inactivation of the transcription factor. The inhibitioncaused by the transcription factor will be removed and transcription andtranslation will occur. Thus a detectable protein will be made and asignal will be received.

[0802] The transcription dimerization assay can be used for any proteinfunction that involves a protein-protein interaction, protein-ligandinteraction and protein-drug interaction. Thus any assay involving suchinteractions can be carried out in the minicell.

[0803] Another non-limiting functional screening assay involves the useof enzymatic function to screen for functionality. In this modality thereceptor or other protein performs a specific enzymatic function. Thisfunction is then carried out in the minicell and monitored usingbiochemical and other techniques. For example if the target protein wasa protease then fluorescent peptides with the cleavage site of theprotease could be used to monitor the activity of the protease. If theprotease was functioning then the peptide would be cleaved and thefluorescents would change. Thus agents can be screened against theprotease in the minicell system and the fluorescents can be monitoredusing specific detection systems. In another non-limiting example, amembrane-bound enzyme such as sphingomyelinase could be expressed inminicells and the minicell particles adsorbed to a standard substratesuch as a 96 well plate. The enzymatic activity could be assessed by astandard in vitro assay involving the release of product(phosphocholine) (e.g., Amplex™ kit A-12220 sold by Molecular Probes).Sphingomyelinase inhibitors could be screened by measuring the reductionof phosphocholine production in the well when presented with substrate(sphingomyelin) in a coupled fluorescence assay.

[0804] Another non-limiting example of minicells used for functionalassays involves the screening of agonists/antagonists for ion channels.In this example the calcium channel, SCaMPER, is encoded on apoycistronic episomal plasmid, which also encodes for a luminescentsoluble protein, aequorin. In this assay, the minicell will containaequorin proteins in its cytoplasm and SCaMPER proteins expressed on theminicell membrane. Thus upon activation of SCaMPER by its ligand, SPC,or by an analog thereof, calcium will flow into the minicell and will bebound by the aequorin which will luminescence. Thus a detection signalfor the functional activation of the calcium channel is obtained.

[0805] Minicell can also be employed for expression of target proteinsand the preparation of membrane preparations for use in screeningassays. Such proteins include but are not limited to receptors (e.g.,GPCRs, sphingolipid receptors, neurotransmitter receptors, sensoryreceptors, growth factor receptors, hormone receptors, chemokinereceptors, cytokine receptors, immunological receptors, and complimentreceptors, FC receptors), channels (e.g., potassium channels, sodiumchannels, calcium channels.), pores (e.g., nuclear pore proteins, waterchannels), ion and other pumps (e.g., calcium pumps, proton pumps),exchangers (e.g., sodium/potassium exchangers, sodium/hydrogenexchangers, potassium/hydrogen exchangers), electron transport proteins(e.g., cytochrome oxidase), enzymes and kinases (e.g., protein kinases,ATPases, GTPases, phosphatases, proteases.), structural/linker proteins(e.g., Caveolins, clathrin), adapter proteins (e.g., TRAD, TRAP, FAN),chemotactic/adhesion proteins (e.g., ICAM11, selecting, CD34, VCAM-1,LFA-1,VLA-1), and chimeric/fusion proteins (e.g., proteins in which anormally soluble protein is attached to a transmembrane region ofanother protein). In such assays the membrane preparations are used toscreen for agents that are either antagonists or agonists. These assaysuse various formats including but not limited to competitive inhibition.

[0806] The format for the screening of minicells includes but is notlimited to the use of test tubes, 6 well plates, 12 well plates, 24 wellplates, 96 well plates, 384 well plates, 1536 well plates, and othermicrotiter well plates. In these systems the minicells can beimmobilized, attached, bound, or fused with the above test tubes orplates. The minicells can also be free in solution for use in tubes andplates. The detection systems for the minicell assay include but are notlimited to fluorescent plate readers, scintillation counters,spectrophotometers, Viewlux CCD Imager, Luminex, ALPHAQuest, BIAcore,FLIPR and F-MAT. Minicell assays can be carried out with but not limitedto techniques such as manual handling, liquid handlers, roboticautomated systems and other formats.

[0807] XVI. C. Chemical Libraries Developments in combinatorialchemistry allow the rapid and economical synthesis of hundreds tothousands of discrete compounds. These compounds are typically arrayedin moderate-sized libraries of small organic molecules designed forefficient screening. Combinatorial methods, can be used to generateunbiased libraries suitable for the identification of novel inhibitors.In addition, smaller, less diverse libraries can be generated that aredescended from a single parent compound with a previously determinedbiological activity. In either case, the lack of efficient screeningsystems to specifically target therapeutically relevant biologicalmolecules produced by combinational chemistry such as inhibitors ofimportant enzymes hampers the optimal use of these resources.

[0808] A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks,” such asreagents. For example, a linear combinatorial chemical library, such asa polypeptide library, is formed by combining a set of chemical buildingblocks (amino acids) in a large number of combinations, and potentaillyin every possible way, for a given compound length (i.e., the number ofamino acids in a polypeptide compound). Millions of chemical compoundscan be synthesized through such combinatorial mixing of chemicalbuilding blocks.

[0809] A “library” may comprise from 2 to 50,000,000 diverse membercompounds. Preferably, a library comprises at least 48 diversecompounds, preferably 96 or more diverse compounds, more preferably 384or more diverse compounds, more preferably, 10,000 or more diversecompounds, preferably more than 100,000 diverse members and mostpreferably more than 1,000,000 diverse member compounds. By “diverse” itis meant that greater than 50% of the compounds in a library havechemical structures that are not identical to any other member of thelibrary. Preferably, greater than 75% of the compounds in a library havechemical structures that are not identical to any other member of thecollection, more preferably greater than 90% and most preferably greaterthan about 99%.

[0810] The preparation of combinatorial chemical libraries is well knownto those of skill in the art. For reviews, see Thompson et al.,Synthesis and application of small molecule libraries, Chem Rev96:555-600, 1996; Kenan et al., Exploring molecular diversity withcombinatorial shape libraries, Trends Biochem Sci 19:57-64, 1994; Janda,Tagged versus untagged libraries: methods for the generation andscreening of combinatorial chemical libraries, Proc Natl Acad Sci U S A.91:10779-85, 1994; Lebl et al., One-bead-one-structure combinatoriallibraries, Biopolymers 37:177-98, 1995; Eichler et al., Peptide,peptidomimetic, and organic synthetic combinatorial libraries, Med ResRev. 15:481-96, 1995; Chabala, Solid-phase combinatorial chemistry andnovel tagging methods for identifying leads, Curr Opin Biotechnol.6:632-9, 1995; Dolle, Discovery of enzyme inhibitors throughcombinatorial chemistry, Mol Divers. 2:223-36, 1997; Fauchere et al.,Peptide and nonpeptide lead discovery using robotically synthesizedsoluble libraries, Can J Physiol Pharmacol. 75:683-9, 1997; Eichler etal., Generation and utilization of synthetic combinatorial libraries,Mol Med Today 1: 174-80, 1995; and Kay et al., Identification of enzymeinhibitors from phage-displayed combinatorial peptide libraries, CombChem High Throughput Screen 4:535-43, 2001.

[0811] Such combinatorial chemical libraries include, but are notlimited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175,Furka, Int. J. Pept. Prot. Res., 37:487-493 (1991) and Houghton, et al.,Nature, 354:84-88 1991). Other chemistries for generating chemicaldiversity libraries can also be used. Such chemistries include, but arenot limited to, peptoids (PCT Publication No. WO 91/19735); encodedpeptides (PCT Publication WO 93/20242); random bio-oligomers (PCTPublication No. WO 92/00091); benzodiazepines (U.S. Pat. No. 5,288,514);diversomers, such as hydantoins, benzodiazepines and dipeptides (Hobbs,et al., Proc. Nat. Acad. Sci. USA, 90:6909-6913 1993); vinylogouspolypeptides (Hagihara, et al., J. Amer. Chem. Soc. 114:6568 1992);nonpeptidal peptidomimetics with .beta.-D-glucose scaffolding(Hirschmann, et al., J. Amer. Chem. Soc., 114:9217-9218 1992); analogousorganic syntheses of small compound libraries (Chen, et al., J. Amer.Chem. Soc., 116:2661 1994); oligocarbamates (Cho, et al., Science,261:1303 1993); and/or peptidyl phosphonates (Campbell, et al., J. Org.Chem. 59:658 1994); nucleic acid libraries (see, Ausubel, Berger andSambrook, all supra); peptide nucleic acid libraries (see, e.g., U.S.Pat. No. 5,539,083); antibody libraries (see, e.g., Vaughn, et al.,Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287);carbohydrate libraries (see, e.g., Liang, et al., Science, 274:1520-1522(1996) and U.S. Pat. No. 5,593,853); small organic molecule libraries(see, e.g., benzodiazepines, Baum C&E News, January 18, page 33 (1993);isoprenoids (U.S. Pat. No. 5,569,588); thiazolidinones andmetathiazanones (U.S. Pat. No. 5,549,974); pyrrolidines (U.S. Pat. Nos.5,525,735 and 5,519,134); morpholino compounds (U.S. Pat. No.5,506,337); benzodiazepines (U.S. Pat. No. 5,288,514); and the like.

[0812] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem.Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd., Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Bio sciences, Columbia, Md., etc.).

[0813] XVI.D. Measuring Enzymatic and Binding Reactions During ScreeningAssays

[0814] Techniques for measuring the progression of enzymatic and bindingreactions in multicontainer carriers are known in the art and include,but are not limited to, the following.

[0815] Spectrophotometric and spectrofluorometric assays are well knownin the art. Examples of such assays include the use of calorimetricassays for the detection of peroxides, as disclosed in Example 1(b) andGordon, A. J. and Ford, R. A., The Chemist's Companion: A Handbook OfPractical Data, Techniques, And References, John Wiley and Sons, N.Y.,1972, Page 437.

[0816] Fluorescence spectrometry may be used to monitor the generationof reaction products. Fluorescence methodology is generally moresensitive than the absorption methodology. The use of fluorescent probesis well known to those skilled in the art. For reviews, see Bashford etal., Spectrophotometry and Spectrofluorometry: A Practical Approach, pp.91-114, IRL Press Ltd. (1987); and Bell, Spectroscopy In Biochemistry,Vol. I, pp. 155-194, CRC Press (1981).

[0817] In spectrofluorometric methods, enzymes are exposed to substratesthat change their intrinsic fluorescence when processed by the targetenzyme. Typically, the substrate is nonfluorescent and converted to afluorophore through one or more reactions. As a non-limiting example,SMase activity can be detected using the Amplex® Red reagent (MolecularProbes, Eugene, Oreg.). In order to measure sphingomyelinase activityusing Amplex Red, the following reactions occur. First, SMase hydrolyzessphingomyelin to yield ceramide and phosphorylcholine. Second, alkalinephosphatase hydrolyzes phosphorylcholine to yield choline. Third,choline is oxidized by choline oxidase to betaine. Finally, H₂O₂, in thepresence of horseradish peroxidase, reacts with Amplex Red to producethe fluorescent product, Resorufin, and the signal therefrom is detectedusing spectrofluorometry.

[0818] Fluorescence polarization (FP) is based on a decrease in thespeed of molecular rotation of a fluorophore that occurs upon binding toa larger molecule, such as a receptor protein, allowing for polarizedfluorescent emission by the bound ligand. FP is empirically determinedby measuring the vertical and horizontal components of fluorophoreemission following excitation with plane polarized light. Polarizedemission is increased when the molecular rotation of a fluorophore isreduced. A fluorophore produces a larger polarized signal when it isbound to a larger molecule (i.e. a receptor), slowing molecular rotationof the fluorophore. The magnitude of the polarized signal relatesquantitatively to the extent of fluorescent ligand binding. Accordingly,polarization of the “bound” signal depends on maintenance of highaffinity binding.

[0819] FP is a homogeneous technology and reactions are very rapid,taking seconds to minutes to reach equilibrium. The reagents are stable,and large batches may be prepared, resulting in high reproducibility.Because of these properties, FP has proven to be highly automatable,often performed with a single incubation with a single, premixed,tracer-receptor reagent. For a eview, see Owickiet al., Application ofFluorescence Polarization Assays in High-Throughput Screening, GeneticEngineering News, 17:27, 1997.

[0820] FP is particularly desirable since its readout is independent ofthe emission intensity (Checovich, W. J., et al., Nature 375:254-256,1995; Dandliker, W. B., et al., Methods in Enzymology 74:3-28, 1981) andis thus insensitive to the presence of colored compounds that quenchfluorescence emission. FP and FRET (see below) are well-suited foridentifying compounds that block interactions between receptors andtheir ligands. See, for example, Parker et al., Development of highthroughput screening assays using fluorescence polarization: nuclearreceptor-ligand-binding and kinase/phosphatase assays, J Biomol Screen5:77-88, 2000.

[0821] Exemplary normal-and-polarized fluorescence readers include thePOLARION fluorescence polarization system (Tecan AG, Hombrechtikon,Switzerland). General multiwell plate readers for other assays areavailable, such as the VERSAMAX reader and the SPECTRAMAX multiwellplate spectrophotometer (both from Molecular Devices).

[0822] Fluorescence resonance energy transfer (FRET) is another usefulassay for detecting interaction and has been described previously. See,e.g., Heim et al., Curr. Biol. 6:178-182, 1996; Mitra et al., Gene173:13-17 1996; and Selvin et al., Meth. Enzymol. 246:300-345, 1995.FRET detects the transfer of energy between two fluorescent substancesin close proximity, having known excitation and emission wavelengths. Asan example, a protein can be expressed as a fusion protein with greenfluorescent protein (GFP). When two fluorescent proteins are inproximity, such as when a protein specifically interacts with a targetmolecule, the resonance energy can be transferred from one excitedmolecule to the other. As a result, the emission spectrum of the sampleshifts, which can be measured by a fluorometer, such as a FMAX multiwellfluorometer (Molecular Devices, Sunnyvale Calif.).

[0823] Scintillation proximity assay (SPA) is a particularly usefulassay for detecting an interaction with the target molecule. SPA iswidely used in the pharmaceutical industry and has been described(Hanselman et al., J. Lipid Res. 38:2365-2373 (1997); Kahl et al., Anal.Biochem. 243:282-283 (1996); Undenfriend et al., Anal. Biochem.161:494-500 (1987)). See also U.S. Pat. Nos. 4,626,513 and 4,568,649,and European Patent No. 0,154,734. An exemplary commercially availablesystem uses FLASHPLATE scintillant-coated plates (NEN Life ScienceProducts, Boston, Mass.).

[0824] The target molecule can be bound to the scintillator plates by avariety of well known means. Scintillant plates are available that arederivatized to bind to fusion proteins such as GST, His6 or Flag fusionproteins. Where the target molecule is a protein complex or a multimer,one protein or subunit can be attached to the plate first, then theother components of the complex added later under binding conditions,resulting in a bound complex.

[0825] In a typical SPA assay, the gene products in the expression poolwill have been radiolabeled and added to the wells, and allowed tointeract with the solid phase, which is the immobilized target moleculeand scintillant coating in the wells. The assay can be measuredimmediately or allowed to reach equilibrium. Either way, when aradiolabel becomes sufficiently close to the scintillant coating, itproduces a signal detectable by a device such as a TOPCOUNT NXTmicroplate scintillation counter (Packard BioScience Co., MeridenConn.). If a radiolabeled expression product binds to the targetmolecule, the radiolabel remains in proximity to the scintillant longenough to produce a detectable signal.

[0826] In contrast, the labeled proteins that do not bind to the targetmolecule, or bind only briefly, will not remain near the scintillantlong enough to produce a signal above background. Any time spent nearthe scintillant caused by random Brownian motion will also not result ina significant amount of signal. Likewise, residual unincorporatedradiolabel used during the expression step may be present, but will notgenerate significant signal because it will be in solution rather thaninteracting with the target molecule. These non-binding interactionswill therefore cause a certain level of background signal that can bemathematically removed. If too many signals are obtained, salt or othermodifiers can be added directly to the assay plates until the desiredspecificity is obtained (Nichols et al., Anal. Biochem. 257:112-119,1998).

[0827] XVI.E. Screening for Novel Antibiotics

[0828] As bacteria and other pathogens acquire resistance to knownantibiotics, there is an ongoing interest in identifying novelantibiotics. See, e.g., Powell W A, Catranis C M, Maynard C A. Syntheticantimicrobial peptide design. Mol Plant Microbe InteractSeptember-October; 1995 8(5):792-4. Minicells can be used to assay,identify and purify novel antibiotics to eubacteria. By way ofnon-limiting example, a minicell that comprises a detectable compoundcan be contacted with a candidate antibiotic to see if the n minicell islysed by a candidate compound, which would release the detectablecompound from the interior of the minicell into solution, this producinga signal that indicates that the candidate antibiotic is effective atlysing bacteria. In such assays, the detectable compound is such that itproduces less or more of the same signal, or a different signal, insidethe minicell as compared to in solution post-lysis. By way ofnon-limiting example, the minicell could comprise a fluorescentcompounds that, when contacted with a second fluorescent compound insolution, produces FRET.

[0829] XVI.F. Reverse Screening

[0830] In one version of minicell display, the invention providesmethods for screening libraries of minicells in which each minicellcomprises an expression element that encodes a few, preferably one,membrane proteins in order to identify a membrane protein that interactswith a preselected compound. By way of non-limiting example, sequencesencoding membrane proteins, fusion proteins, or cytoplasmic proteins arecloned into an expression vector, either by “shotgun” cloning or bydirected cloning, e.g., by screening or selecting for cDNA clones, or byPCR amplification of DNA fragments, that encode a protein using one ormore oligonucleotides encoding a highly conserved region of a proteinfamily. For a non-limiting example of such techniques, see Krautwurst,D., et al. 1998. Identification of ligands for olfactory receptors byfunctional expression of a receptor library. Cell 95:917-926. By way ofnon-limiting example, a minicell expressing a receptor binds apreselected ligand, which may be a drug. Various assays for receptorbinding, enzymatic activity, and channeling events are known in the artand may include detectable compounds; in the case of binding assays,competition assays may also be used (Masimirembwa, C. M., et al. 2001.In vitro high throughput screening of compounds for favorable metabolicproperties in drug discovery. Comb. Chem. High Throughput Screen.4:245-263; Mattheakis, L. C., and A. Saychenko. 2001. Assay technologiesfor screening ion channel targets. Curr. Opin. Drug Discov. Devel.4:124-134; Numann, R., and P. A. Negulescu. 2001. High-throughputscreening strategies for cardiac ion channels. Trends Cardiovasc. Med.11:54-59; Le Poul, E., et al. 2002. Adaptation of aequorin functionalassay to high throughput screening. J. Biomol. Screen. 7:57-65; andGraham, D. L., et al. 2001. Application of beta-galactosidase enzymecomplementation technology as a high throughput screening format forantagonists of the epidermal growth factor receptor. J. Biomol. Screen.6:401-411).

[0831] Once a minicell has been identified by an assay and isolated, DNAis prepared from the minicell. The cloned DNA present in the minicellencodes the receptor displayed by the minicell. Having been cloned, thereceptor is used as a therapeutic target. For example, the receptor isproduced via recombinant DNA expression and used in minicell-based orother assays to identify and characterize known and novel compounds thatare ligands, antagonists and/or agonists of the cloned receptor. Theligands, antagonists and agonists may be used as lead compounds and/ordrugs to treat diseases in which the receptor plays a role. Inparticular, when the preselected ligand is a drug, diseases for whichthat drug is therapeutic are expected to be treated using the novelligands, antagonists and agonists, or drugs and prodrugs developedtherefrom.

[0832] Preparations of minicells that express and secrete secretes asoluble protein can be prepared in order to identify ligands, includingbut not limited to small molecules, that interact with the solubleprotein. Soluble proteins include, but are not limited to, knownsecreted or proteolytically cleaved proteins and peptides, hormones andcytokines. In this format, minicells are placed in, or adhered to, thewells of a microtiter multiwell plate. A different compound or group ofcompounds is placed in each well, along with any reagents necessary togenerate or squelch a signal corresponding to a change in the solubleprotein produced by the minicell. Such changes include, by way ofnon-limiting example, conformational changes in the protein that mayoccur as a result of binding of a ligand or otherwise. A well thatgenerates the appropriate signal contains a compound that causes achange in the soluble protein.

[0833] It is also possible to carry out procedures such as the onedescribed in the immediately preceding paragraph “in reverse.” In thisformat, a known ligand, which may be a drug, is used to identify solubleproteins that bind to the ligand/drug. Libraries of minicells whereineach minicell secretes a different soluble protein are prepared, andeach type of minicell is placed into, or adhered to the wall of, a wellof a microtiter plate, along with reagents for generating a signal whenthe ligand/drug binds to a soluble protein. Minicells that generate theappropriate signal comprise a cloned DNA that encodes a soluble proteinthat interacts with the known ligand/drug. Once cloned, the solubleprotein is prepared and used as a therapeutic target in order toidentify known or novel compounds that bind thereto. When thepreselected ligand is a drug, diseases for which that drug istherapeutic are expected to be treated using the known and novelcompounds so identified, or using drugs and prodrugs developed from suchcompounds.

[0834] Mincells expressing known membrane and soluble proteins can alsobe used to help characterize lead compounds and accelerate thegeneration of drugs therefrom. In particular, such studies may be usedidentify potentially detrimental interactions that might occur upon invivo administration, e.g., ADME/Tox screening (Ekins, S., et al. 2002.In silico ADME/Tox: the state of the art. J. Mol. Graph. Model.20:305-309; and Li, A., et al. 2002. Early ADME/Tox studies and insilico screening. Drug Discov. Today 7:25-27).

[0835] By way of non-limiting example, a human receptor that is known tobe important for the normal functioning of a cell may be expressed inmincells, and various chemical derivatives of a lead compound can betested to ensure that they do not bind to the receptor, as such bindingwould be expected to have adverse effects in vivo. As another example,an enzyme that degrades a drug, such as a cytochrome P450, is expressedin mincells and used to examine the susceptibility of a candidate drugto such degradation. The cytochrome P450 family of enzymes is primarilyresponsible for the metabolism of xenobiotics such as drugs, carcinogensand environmental chemicals, as well as several classes of endobioticssuch as steroids and prostaglandins. Exemplary P450 cytochromes involvedin drug degradation include, but are not limited to, CYP2D6 (cytochromeP4502D6, also known as debrisoquine hydroxylase), CYP1A1, CYP1A2,CYP2A6, CYP2B6, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4 andCYP3A5.

[0836] XVI.G. Molecular Variants

[0837] In one aspect of the invention, minicells are used in methods ofscreening to identify agents that improve, enhance, or decrease theinteraction of a protein with another compound. These methods include,by way of non-limiting example, modification of protein targets throughdirected or random mutagenic approaches to identify criticalinteractions between a wild-type protein target and a specific drugmolecule. Information obtained from studies of mutant proteins is usedto specifically produce or modify a therapeutic agent to interact morespecifically and/or effectively with the wild-type protein target, thusincreasing the therapeutic efficacy of the parental drug and/ordecreasing non-specific, potentially deleterious interactions. See, forexample, Lietha, D., et al. 2001. Crystal structures of NK1-heparincomplexes reveal the basis for NK1 activity and enable engineering ofpotent agonists of the MET receptor. EMBO J. 20:5543-5555; and Chen, Y.Z., et al. Can an optimization/scoring procedure in ligand-proteindocking be employed to probe drug-resistant mutations in proteins? J.Mol. Graph. Model. 19:560-570; Zhao, H. and F. H. Arnold. Combinatorialprotein design: Strategies for screening protein libraries. CurrentOpinion in Structural Biology 7:480-485 (1997); and Carrupt P A, elTayar N, Karlen A, Testa B. Molecular electrostatic potentials forcharacterizing drug-biosystem interactions. Methods Enzymol.1991;203:638-77. Martin Y C. Computer-assisted rational drug design.Methods Enzymol. 1991;203:587-613.

[0838] By way of non-limiting example, information obtained using themethods of the invention may be in conjunction with x-raycrystallographic structural determinations to characterizereceptor:ligand interactions (Muller, G. 2000. Towards 3D structures ofG protein-coupled receptors: a multidisciplinary approach. Curr. Med.Chem. 7:861-888). By way of non-limiting example, minicells may be usedto display the family of molecular variants to characterize the specificmutagenic changes on the functional properties of the protein.

[0839] Studies of variant proteins ran also be used to modify drugs tofit natural variants of proteins, especially those associated withpathogens. Pathogens such as viruses, including retroviruses such asHIV, may acquire mutations that change a site where a drug acts, therebyrendering the pathogen immune to the drug. Studies of variant proteinscan be used to quickly produce derivatives of a drug that are activeagainst a variant protein. See, for example, Varghese J N, Smith P W,Sollis S L, Blick T J, Sahasrabudhe A, McKimm-Breschkin J L, Colman P M.Drug design against a shifting target: a structural basis for resistanceto inhibitors in a variant of influenza virus neuraminidase. StructureJun. 15 1998;6(6):735-46; and Baldwin E T, Bhat T N, Liu B, PattabiramanN, Erickson J W. Structural basis of drug resistance for the V82A mutantof HIV-1 proteinase. 78: Nat Struct Biol March 1995;2(3):244-

[0840] XVI.H. Directed Evolution

[0841] The minicells and methods described herein can be used indirected evolution. Unlike natural variation, directed evolutiongenerates new protein variants in vitro (see, e.g., Arnold, F. H. and A.A. Violkov. Directed Evolution of Biocatalysts. Curr Op Chem Biol 1999.3:54-59). Amino acid substitutions can be introduced into a protein ofinterest by mutating the gene encoding the protein. Mutations areintroduced by, e.g., replicating DNA in mutator strains, by chemicalmutagenesis or radiation-induced mutagenesis (Drake, J. W., TheMolecular Basis of Mutation, Holden-Day, San Francisco, 1970). Othermethods include error-prone PCR and “domain shuffling” (Moore, G. L. andC. D. Maranas. Modeling DNA Mutation and Recombination for DirectedEvolution Experiments. J. Ther. Biol. 2000. 205:483-503). In the lattermethod, different regions of members of the same gene family arerecombined so that the inherent variability of members of the family isused to produce novel “isoforms” of genes.

[0842] A group of variants is screened to select for those variantswhich have the desired activity. The activity of the initial variantsthat are so isolated may be inadequate for a given application, but theprocess can be repeated using these initial members to generate a secondgroup of variants, or reiterated as many times as is necessary toproduce one or more variants having the desired activity orcharacteristics.

[0843] XVI.I. Isolation and Characterization of Components of SignalTranduction Pathways

[0844] In one version of minicell display, the invention providesmethods for screening libraries of minicells, in which each minicellcomprises a preselected component of a signal transduction pathway, inorder to identify soluble and membrane proteins that interact with thepreselected component. By way of non-limiting example, a plurality ofminicells, each of which displays the same G-protein-coupled receptor(GPCR), is used to prepare a minicell library in which a differentG-protein encoding sequence is present and expressed in each minicell.Minicells comprising a G-protein that interacts with the GPCR areidentified, e.g., via transactivation assays described in Example 18.Once a minicell has been identified by an assay and isolated, DNA isprepared from the minicell. The cloned DNA present in the minicellencodes a G-protein that interacts with the GPCR of the dsiplayed by theminicells of the library. Having been cloned, the G-protein is used as atherapeutic target that can be used in screening assays to identifynovel lead compounds and drugs that interfere or alter the activity ofthe GPCR. In particular, when the GPCR of the minicell library is knownto be a therapeutic target for a specific disease, it is expected thatcompounds that interfere or alter the activity of a G-protein thatinteracts with the GPCR will be or lead to therapeutics for thatspecific disease.

[0845] In addition to G-protein signal transduction pathways, othernon-limiting examples of signal tranduction pathways include the MAPKpathway, the SAPK pathway, the p38 pathway and/or the ceramide-mediatedstress response pathway. See Zhang, W., and L. E. Samelson. 2000. Therole of membrane-associated adaptors in T cell receptor signalling.Semin. Immunol. 12:35-41; Liebmann, C. 2001. Regulation of MAP kinaseactivity by peptide receptor signalling pathway: paradigms ofmultiplicity. Cell Signal. 13:777-785; Lee, Jr., J. T., and J. A.McCubrey. 2002. The Raf/MEK/ERK signal transduction cascade as a targetfor chemotherapeutic intervention in leukemia. Leukemia. 16:486-507;Tibbles, L. A., and J. R. Woodgett. 1999. The stress-activated proteinkinase pathways. Cell Mol. Life Sci. 55:1230-1254; Rao, K. M. 2001. MAPkinase activation of macrophages. J. Leukoc. Biol. 69:3-10; Pelech, S.L., and D. L. Charest. 1995. MAP kinase-dependent pathways in cell cyclecontrol. Prog. Cell Cycle Res. 1:33-52; Lee, S. H., et al. 2001.BetaPix-enhanced p38 activation by Cdc42/Rac/PAK/MKK3/6-mediatedpathway. Implication in the regulation of membrane ruffling. J. Biol.Chem. 276:25066-25072; Ono, K., et al. 2000. The p38 signal transductionpathway Activation and function. Cellular Signalling 12:1-13; You, A.2001. Differentiation, apoptosis, and function of human immature andmature myeloid cells: intracellular signaling mechanism. Int. J.Hematol. 73:438-452; Johnson, D. I. 1999. Cdc42: An essential Rho-typeGTPase controlling eukaryotic cell polarity. Microbiol. Mol. Biol. Rev.63:54-105; Williams, J. A. 2001. Intracellular signaling mechanismsactivated by cholecystokinin-regulating synthesis and secretion ofdigestive enzymes in pancreatic acinar cells. Annu. Rev. Physiol.63:77-97; Mathias, S., et al. 1998. Signal trasduction of stress viaceramide. Biocehm J. 335:465-480; and Hannun, Y. A., et al. 2000.Ceramide in the eukaryotic stress response. Trends Cell Biol. 10:73-80.

XVII. Determinating the Structures of Membrane Proteins

[0846] Three-dimensional (3D) structures of proteins may be used fordrug discovery. However, GPCRs and other membrane proteins presentchallenging problems for 3D structure determination. Muller, Towards 3Dstructures of G protein-coupled receptors: a multidisciplinary approach.(Review), Curr Med Chem 2000 pp.861-88; Levy et al., Two-dimensionalcrystallization on lipid layer: A successful approach for membraneproteins, J Struct Biol 1999 127, 44-52. Although the three-dimensionalstructures of hundreds of different folds of globular proteins have beendetermined, fewer than 20 different integral membrane protein structureshave been determined. There are many reasons for this. Extractingmembrane proteins from the membrane can easily disrupt their nativestructure, and membrane proteins are notoriously difficult tocrystallize.

[0847] Some membrane proteins readily form two-dimensional crystals inmembranes and can be used for structure determination using electrondiffraction spectroscopy (ED) instead of x-ray crystallography. This isthe technique that was used to determine the structure ofbacteriorhodopsin (see below).

[0848] Nuclear magnetic resonance (NMR) is an alternative method fordetermining membrane protein structure, but most membrane proteins aretoo large for high-resolution NMR at the present state of the art.Furthermore, membrane proteins require special conditions for NMR, e.g.deuterated lipids must be used to avoid confusing the signal of theprotein protons with the noise of membrane lipid protons.

[0849] Membrane protein for which structures have been determinedinclude photosynthetic reaction center, porin, porin OmpF, plantlight-harvesting complex (chlorophyll a-b binding protein), bacteriallight-harvesting complex, cytochrome c oxidase, glycophorin A, the Sec Atranslocation ATPase of Bacillus subtilis, and a bacterial potassiumchannel. For details, see: Weinkauf et al., (2001): Conformationalstabilization and crystallization of the Sec A translocation ATPase fromBacillus subtilis. Acta Crystallogr D Biol Crystallogr 57:559-565; Cowanet al., (1992): Crystal structures explain functional properties of twoE. coli porins. Nature 358:727-33; Deisenhofer et al., (1984): X-raystructure analysis of a membrane protein complex. Electron density mapat 3 A resolution and a model of the chromophores of the photosyntheticreaction center from Rhodopseudomonas viridis. J Mol Biol 180:385-98;Deisenhofer et al., (1985): Structure of the protein subunits in thephotosynthetic reaction centre of Rhodopseudomonas viridis at 3Angstroms resolution. Nature 318:618; Doyle et al., (1998): Thestructure of the potassium channel: molecular basis of K+conduction andselectivity. Science 280:69-77; Henderson et al., (1990): Model for thestructure of bacteriorhodopsin based on high-resolution electroncryo-microscopy. J Mol Biol 213:899-929; Iwata et al., (1998): Completestructure of the 11-subunit bovine mitochondrial cytochrome bc Icomplex. Science 281:64-71; Koepke et al., (1996): The crystal structureof the light-harvesting complex II (B800-850) from Rhodospirillummolischianum. Structure 4:581-97; Kuhlbrandt et al., (1994): Atomicmodel of plant light-harvesting complex by electron crystallography.Nature 367:614-21; Lemmon et al., (1992): Sequence specificity in thedimerization of transmembrane alpha-helices. Biochemistry 31:12719-25;MacKenzie et al., (1997): A transmembrane helix dimer: structure andimplications. Science 276:131-3; McDermott et al., (1995): Crystalstructure of an integral membrane light-harvesting complex fromphotosynthetic bacteria, Nature 374:517-21; Michel (1982):Three-dimensional crystals of a membrane protein complex. Thephotosynthetic reaction centre from Rhodopseudomonas viridis. J Mol Biol158:567-72; Tsukihara et al., (1996): The whole structure of the13-subunit oxidized cytochrome c oxidase at 2.8 A. Science 272:1136-44;and Weiss et al., (1991): The structure of porin from Rhodobactercapsulatus at 1.8 A resolution. FEBS Lett 280:379-82. Table 5, which isbased upon Preusch et al. (1998) as revised by White & Wimley (1999),lists membrane proteins whose crystallographic structures have beendetermined. TABLE 5 Structural Data Regarding Membrane Proteins PROTEINREFERENCES MONOTOPIC MEMBRANE PROTEINS Portaglandin H2 synthase-1.Sheep. 3.5 Å Picot et al. (1994) Cyclooxygenase-2. Mus Musculus. 3.0 ÅKurumbail et al. (1996) Squalene-hopene cyclase. Alicyclobacillusacidocaldarius. Wendt et al. (1999) 2.0 Å TRANSMEMBRANE PROTEINSBacterial Rhodopsins (Halobacterium salinarium) Bacteriorhodopsin (BR)2D xtals. EM. 3.5 Å Grigrorieff et al. (1996) 2D xtals. EM. 3.0 Å Kimuraet al. (1997) 3D xtals. X-ray. 2.5 Å Pebay-Peyroula et al. (1997) 3Dxtals. X-ray. 1.9 Å Belhrhali et al. (1999) 3D xtals. X-ray 2.1 Å Kintermediate Edman et al. (1999) 3D xtals. X-ray. 2.3 Å Luecke et al.(1998) 3D xtals. X-ray. 1.55 Å Luecke et al. (1999) 3D xtals. X-ray.D96N mutant (BR) 1.80 Å. Luecke et al. (1999) 3D xtals. X-ray. D96Nmutant (M) 2.00 Å 3D xtals. X-ray. 2.9 Å Essen et al. (1998)Halorhodopsin (HR) 3D xtals. Xray. 1.8 Å Kolbe et al. (2000) GPROTEIN-COUPLED RECEPTORS Rhodopsin. Bovine Rod Outer Segment. 2.8 ÅPalczewski et al. (2000) Photosynthetic Reaction CentersRhodopseudomonas virdis. 2.3 Å Deisenhofer et al. (1985) Rhodobactersphaeroides. 3.0 Å Yeates et al. (1987) Rhodobacter sphaeroides. 3.1 ÅChang et al. (1991) Light Harvesting Complexes Rhodopseudomonasacidophila. 2.5 Å McDermott et al. (1995) Rhodospirillum molischianum.2.4 Å Koepke et al. (1996) Photosystems Photosystem I. Synechococcuselongates 4.0 Å Schubert et al. (1997) Photosystem II. Synechocoocuselongates 3.8 Å Zouni et al. (2001) Beta-Barrel MembraneProteins-Multimeric (Porins and Relatives) Porin. Rhodobactercapsulatus. 1.8 Å Weiss & Schulz (1992) Porin. Rhodopeudomonas blastica1.96 Å Kreutsch et al. (1994) OmpF. E. coli. 2.4 Å Cowan et al. (1992)PhoE. E. coli. 3.0 Å Cowan et al. (1992) Maltoporin. Salmonellatyphimurium. 2.4 Å Meyer et al. (1997) Maltoporin. E. coli 3.1 ÅSchirmer et al. (1995) Beta-Barrel Membrane Proteins-Monomeric/DimericTolC outer membrane protein. E. coli 2.1 Å Protein is a Koronakis et al.(2000) trimer, each contributing 4 strands to a single barrel. OmpA. E.coli. 2.5 Å Pautsch & Schulz (1998) OmpA E. coli. By NMR, in DPCmicelles Arora et al. (2001) OmpX. E. coli. 1.9 Å Vogt & Schulz (1990)OMPLA (outer membrane phospholipase A) E. coli. 2.1 Å. Snijder et al.(1999) monomer (1QD5) and dimer (1QD6) FhuA. E. coli. 2.5 Å Ferguson etal. (1998); Lambert et al., 1999 FhuA + ferrichrome-iron. E. coli. 2.7 ÅBuchanan et al. (1999) FepA. E. coli. 2.4 Å Ferguson et al. (1999)Glycophorin A.humanm. MacKenzie et al. (1997) Non-constitutive Toxins,etc. Alpha-hemolysin. Staphylococcus aureus. 1.9 Å Song et al. (1996)LukF. Staphylococcus aureus. 1.9 Å Olson et al. (1999) Ion Channels KcsAPotassium, H⁺ gated. Streptomyces lividans. 3.2 Å Doyle et al. (1998)MscL Mechanosensitive. Mycobacterium tuberculosis. Chang et al. (1998)3.5 Å Other Channels AQP1 - aquaporin water channel. Red blood cell.Murata et al. (2000) Electron crystallography in membrane plane. 3.8 ÅAQP1 - In vitreous ice by electron microscopy. 3.7 Å Ren et al. (2000)GipF - glycerol facilitator channel. E. coli. 2.2 Å Fu et al. (2000)P-type ATPase Calcium ATPase. Sarcoplasmic reticulum. Rabbit. 2.6 ÅToyoshima et al. (2000) Respiratory Proteins Fumerate Reductase Complex.Escherichia coli. 3.3 Å Iverson et al. (1999) Fumerate ReductaseComplex. Wolinella succinogenes Lancaster et al. (1999) 2.2 Å ATPsynthase (F₁c₁₀). S. cerevisiae. 3.9 Å. X-ray Stock et al. (1999)structure is a C alpha model derived from composite of 1BMF, 1A91 & 1AQTCytochrome C Oxidases aa₃ bovine heart mitochondria. 2.8 Å Tsukihara etal. (1996) aa₃ Paracoccus denitrificans. 2.8 Å Iwata et al. (1995) ba₃from T. thermophilus. 2.4 Å Soulimane et al. (2000) Cytochrome bc₁Complexes Bovine Heart Mitochondria (5 subunits). 2.9 Å Xia et al.(1997) Chicken Heart Mitochondria. 3.16 Å Zhang et al. (1998) BovineHeart Mitochondria (11 subunits). 2.8-3.0 Å. Iwata et al. (1998) S.cerevisiae (yeast, 9 subunits). 2.3 Å Hunte et al. (2000)

[0850] Citations for Table 5

[0851] Arora et al., (2001). Structure of outer membrane protein Atransmembrane domain by NMR spectroscopy. Nature Structural Biol. 8,334-338.

[0852] Belrhali et al., (1999). Protein, lipid, and water organizationin bacteriorhodopsin crystals: A molecular view of the purple membraneat 1.9 Å resolution. Structure 7:909-917.

[0853] Buchanan et al., (1999). Crystal Structure of the outer membraneactive transporter FepA from Escherichia coli. Nature Struc. Biol.6:56-63.

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XVIII. Biosensors and Environmental Applications

[0906] XVIII.A. Minicell-Based Biosensors

[0907] The present invention is directed to a device that comprises asensor adapted to detect one or more specific health and/or nutritionmarkers in a subject or in the environment. The device may also signalthe caretaker, the subject, or an actuator of the occurrence. The sensorcomprises a biosensor. As used herein, the term “biosensor” is definedas a component comprising one or more binding moities being adapted todetect a ligand found in one or more target pathogenic microorganisms orrelated biomolecules.

[0908] Generally, biosensors function by providing a means ofspecifically binding, and therefore detecting, a target biologicallyactive analyte. In this way, the biosensor is highly selective, evenwhen presented with a mixture of many chemical and biological entities.Often the target biological analyte is a minor component of a complexmixture comprising a multiplicity of biological and other components.Thus, in many biosensor applications, detection of target analytesoccurs in the parts-per-billion, parts-per-trillion, or even lowerranges levels.

[0909] XVIII.A.1. Minicell-Based Biosensor Design

[0910] The biosensor of the present invention may comprise abio-recognition element, or molecular recognition element, that providesthe highly specific binding or detection selectivity for a particularanalyte. In a biosensor of the invention, the bio-recognition element,or system, is a minicell displaying an enzyme or sequence of enzymes; anantibody or antibody derivative; a membrane receptor protein; or thelike, and generally functions to interact specifically with a targetbiological analyte. The bio-recognition element is responsible for theselective recognition of the analyte and the physico-chemical signalthat provides the basis for the output signal. The expressed protein ormolecule does not need to be a naturally occurring membrane boundprotein but could be a soluble protein or small molecule thethered tothe minicell by, for example, a transmembrane domain of another proteinsuch as the EGFR or ToxR.

[0911] Biosensors may include biocatalytic biosensors, and bioaffmitybiosensors. In biocatalytic biosensor embodiments, the bio-recognitionelement minicell is “biocatalytic,” e.g., displays an enzyme. Inbiocatalytic biosensors, the selective binding sites “turn over” (i.e.,can be used again during the detection process), resulting in asignificant amplification of the input signal. Biocatalytic sensors suchas these are generally useful for real-time, continuous sensing.

[0912] Bioaffinity sensors are generally applicable to bacteria,viruses, toxins and other undesirable compounds and includechemoreceptor-based biosensors and/or immunological sensors (i.e.,immunosensors). Chemoreceptors are complex biomolecular macroassembliesresponsible, in part, for a viable organism's ability to sense chemicalsin its environment with high selectivity. Chemoreceptor-based biosensorscomprise one or more natural or synthetic chemoreceptors associated witha means to provide a signal (visual, electrical, etc.) of the presenceor concentration of a target biological analyte. In certain embodiments,the chemoreceptor may be associated with an electrode (i.e., anelectrical transducer) so as to provide a detectable electrical signal.In the biosensors of the invention, minicells displaying a receptor areused in place of chemoreceptors. The minicell has many desired featuresof a viable cell, and performs similar functions, but is more durable.

[0913] On the other hand, the bio-recognition elements of immunosensorsare generally antibodies or antibody derivatives. In any case,bioaffinity biosensors are generally irreversible because the receptorsites of the biosensor become saturated when exposed to the targetbiological analyte. In a biosensor of the invention, an immunosensor maybe a minicell displaying an antibody or antibody fragment.

[0914] Biocatalytic and bioaffinity biosensor systems are described inmore detail in Journal of Chromatography, 510 (1990) 347-354 and in theKirk-Othmer Encyclopedia of Chemical Technology, 4.sup.th ed. (1992),John Wiley & Sons, NY, the disclosure of which is incorporated byreference herein.

[0915] The biosensors of the present invention may detect biologicallyactive analytes related to impending (i.e., future presentation ofsymptoms is likely) or current human systemic disease states, including,but not limited to, pathogenic bacteria, parasites (e.g., any stage ofthe life cycle, including eggs or portions thereof, cysts, or matureorganisms), viruses, fungi such as Candida albicans, antibodies topathogens, and/or microbially produced toxins. Additionally, thebiosensor may target biologically active analytes related to impendingor current localized health issues, such as stress proteins (e.g.,cytokines) and interleukin 1-alpha that may precede the clinicalpresentation of skin irritation or inflammation. In preferredembodiments, the biosensor functions as a proactive sensor, detectingand signaling the subject, a caretaker or medical personnel of theimpending condition prior to the presentation of clinical symptoms. Thisallows time to administer prophylactic or remedial treatments to thesubject which can significantly reduce, if not prevent, the severity andduration of the symptoms. Further, the sensor, by detecting the presenceof a target biological analyte in a sample from the subject, may detectresidual contamination on a surface, such as skin or environmentalsurface, in contact with the biosensor, and provide and appropriatesignal.

[0916] The physico-chemical signal generated by the bio-recognitionelement or elements may be communicated visually to the caretaker ormedical personnel (i.e., via a color change visible to the human eye).Other embodiments may produce optical signals, which may require otherinstrumentation to enhance the signal. These include flourescence,bioluminesence, total internal reflectance resonance, surface plasmonresonance, Raman methods and other laser-based methods, such as LED orlaser diode sensors. For example, exemplary surface plasmon resonancebiosensors are available as IBIS I and IBIS II from XanTecAnalysensysteme of Muenster, Germany, which may comprise bioconjugatesurfaces as bio-recognition elements. Alternatively, the signal may beprocessed via an associated transducer which, for example, may producean electrical signal (e.g., current, potential, inductance, orimpedance) that may be displayed (e.g., on a readout such as an LED orLCD display) or which triggers an audible or tactile (e.g., vibration)signal or which may trigger an actuator, as described herein. The signalmay be qualitative (e.g., indicating the presence of the targetbiological analyte) or quantitative (i.e., a measurement of the amountor concentration of the target biological analyte). In such embodiments,the transducer may optionally produce an optical, thermal or acousticsignal.

[0917] In any case, the signal may also be durable (i.e., stable andreadable over a length of time typically at least of the same magnitudeas the usage life of the device) or transient (i.e., registering areal-time measurement). Additionally, the signal may be transmitted to aremote indicator site (e.g., via a wire, or transmitter, such as aninfrared or rf transmitter) including other locations within or on thedevice or remote devices. Further, the sensor, or any of its components,may be adapted to detect and/or signal only concentrations of the targetbiological analyte above a predefined threshold level (e.g., in caseswherein the target biological analyte is normally present in the bodilywaste or when the concentration of the analyte is below a known “danger”level).

[0918] The target analytes that the biosensors of the present inventionare adapted to detect may also be viruses. These may includediarrhea-inducing viruses such as rotavirus, or other viruses such asrhinovirus and human immunodeficiency virus (HIV). An exemplarybiosensor adapted to detect HIV is described in U.S. Pat. Nos. 5,830,341and 5,795,453, referenced above. The disclosure of each of these patentsis incorporated by reference herein. Biosensors are adopted to use indifferent tissues; see, e.g., U.S. Pat. No. 6,342,037; Roe et al. Jan.29, 2002; Device having fecal component sensor; and using differentbinding molecules, see, e.g., U.S. Pat. No. 6,329,160; Schneider et al.Dec. 11, 2001; Biosensors.

[0919] When minicells are incorporated into a biosensor, they may beimmobilized in the biosensor by techniques known in the art such asentrapment, adsorption, crosslinking, encapsulation, covalentattachment, any combination thereof, or the like. Further, theimmobilization can be carried out on many different substrates such asknown the art. In certain preferred embodiments, the immobilizationsubstrate may be selected from the group of polymer-based materials,hydrogels, tissues, nonwoven materials or woven materials.

[0920] In certain embodiments, biosensor embodiments, may comprise, bedisposed on, or be operatively associated with a microchip, such as asilicon chip, MEMs (i.e., micro electromechanical system) device, or anintegrated circuit. Microchip-based biosensors may be known as“biochips”. Regardless of the type of sensor, the microchip may comprisea multiplicity of sensor components having similar or differentsensitivities, kinetics, and/or target analytes (i.e., markers) in anarray adapted to detect differing levels or combinations of theanalyte(s). Further, each sensor in such an array may provide adifferent type of signal, including those types disclosed herein, andmay be associated with different actuators and/or controllers. Also,each sensor in an array may operate independently or in association with(e.g., in parallel, combination, or series) any number of other sensorsin the array.

[0921] A minicell of a biosensor of the invention may comprise adetectable compound that produces a signal once ligands have bound tothe minicell. By way of non-limiting example, a minicell may display areceptor for a ligand and contain a fluorescent compound. The bindingand internalization of the ligand into the minicell results in FRET,shifting the wavelength of the signal. See, by way of non-limitingexample, Billinton et al., Development of a green fluorescent proteinreporter for a yeast genotoxicity biosensor, Biosensors & Bioelectronics13:831-838, 1998. A biosensor according to the invention may usemicrobalance sensor systems (Hengerer et al., Determination of phageantibody affinities to antigen by a microbalance sensor system,BioTechniques 26:956-964, 1999).

[0922] XVIII.A.2. Surface Plasmon Resonance

[0923] Kd is measured using surface plasmon resonance on a chip, forexample, with a BIAcore® chip coated with immobilized bindingcomponents, or similar systems such as the IAsys from Thermo Labsystems,Affinity Sensors Division (Cambridge, U.K.) or the BIOS-1 system fromArtificial Sensing, Inc. (Zurich, Switzerland). See Fitzgerald, Couplingoptical biosensor technology with micropreparative HPLC: Part 1, AmBiotech Lab November 2000, p.10 and 12; Fitzgerald, Coupling opticalbiosensor technology with micropreparative HPLC: Part 2, Am Biotech LabFeb. 14, 16 and 18; 2001, and Leatherbarrow et al., Analysis ofmolecualr recognition using optical sensors, Current Opinion in ChemBiol 3:544-547, 1999).

[0924] Surface plasmon resonance is used to characterize the microscopicassociation and dissociation constants of reaction between an antibodyor antibody fragment and its ligand. Such methods are generallydescribed in the following references that are incorporated herein byreference. (Vely F. et al., BIAcore analysis to test phosphopeptide-SH2domain interactions, Methods in Molecular Biology. 121:313-21, 2000;Liparoto et al., Biosensor analysis of the interleukin-2 receptorcomplex, Journal of Molecular Recognition. 12:316-21, 1999; Lipschultzet al., Experimental design for analysis of complex kinetics usingsurface plasmon resonance, Methods. 20):310-8, 2000; Malmqvist.,BIACORE: an affinity biosensor system for characterization ofbiomolecular interactions, Biochemical Society Transactions 27:335-40,1999; Alfthan, Surface plasmon resonance biosensors as a tool inantibody engineering, Biosensors & Bioelectronics. 13:653-63, 1998;Fivash et al., BIAcore for macromolecular interaction, Current Opinionin Biotechnology. 9:97-101, 1998; Price et al.; Summary report on theISOBM TD-4 Workshop: analysis of 56 monoclonal antibodies against theMUC1 mucin. Tumour Biology 19 Suppl 1:1-20, 1998; Malmqvist et al,Biomolecular interaction analysis: affinity biosensor technologies forfunctional analysis of proteins, Current Opinion in Chemical Biology.1:378-83, 1997; O'Shannessy et al., Interpretation of deviations frompseudo-first-order kinetic behavior in the characterization of ligandbinding by biosensor technology, Analytical Biochemistry. 236:275-83,1996; Malmborg et al., BIAcore as a tool in antibody engineering,Journal of Immunological Methods. 183:7-13, 1995; Van Regenmortel, Useof biosensors to characterize recombinant proteins, Developments inBiological Standardization. 83:143-51, 1994; O'Shannessy, Determinationof kinetic rate and equilibrium binding constants for macromolecularinteractions: a critique of the surface plasmon resonance literature,Current Opinions in Biotechnology. 5:65-71, 1994).

[0925] BIAcore® uses the optical properties of surface plasmon resonance(SPR) to detect alterations in protein concentration bound within to adextran matrix lying on the surface of a gold/glass sensor chipinterface, a dextran biosensor matrix. In brief, proteins are covalentlybound to the dextran matrix at a known concentration and a ligand forthe protein (e.g., antibody) is injected through the dextran matrix.Near infra red light, directed onto the opposite side of the sensor chipsurface is reflected and also induces an evanescent wave in the goldfilm, which in turn, causes an intensity dip in the reflected light at aparticular angle known as the resonance angle. If the refractive indexof the sensor chip surface is altered (e.g., by ligand binding to thebound protein) a shift occurs in the resonance angle. This angle shiftcan be measured and is expressed as resonance units (RUs) such that 1000RUs is equivalent to a change in surface protein concentration of 1ng/mm2. These changes are displayed with respect to time along they-axis of a sensorgram, which depicts the association and dissociationof any biological reaction.

[0926] Additional details may be found in Jonsson et al., Introducing abiosensor based technology for real-time biospecific interactionanalysis, (1993) Ann. Biol. Clin. 51:19-26; Jonsson et al., Real-timebiospecific interaction analysis using surface plasmon resonance and asensor chip technology, (1991) Biotechniques 11:620-627; Johnsson etal., Comparison of methods for immobilization to carboxymethyl dextransensor surfaces by analysis of the specific activity of monoclonalantibodies, (1995) J. Mol. Recognit. 8:125-131; and Johnsson,Immobilization of proteins to a carboxymethyldextran-modified goldsurface for biospecific interaction analysis in surface plasmonresonance sensors (1991) Anal. Biochem. 198:268-277, Karlsson et. al.,Kinetic analysis of monoclonal antibody-antigen interactions with a newbiosensor based analytical system J. Immunol. Meth., 145, 229, 1991;Weinberger et al., Recent trends in protein biochip technology,Pharmacogenomics November 2000; 1(4):395-416; Lipschultz et al.,Experimental design for analysis of complex kinetics using surfaceplasmon resonance, Methods March 2000;20(3):310-8.

[0927] XVIII.B. Toxicological Sampling

[0928] Minicells are ideally suited for in vitro diagnostictoxicological applications in which toxins, poisons, infectious agentsor pathogens, heavy metals, pollutants, caustic agents, allergens,organic molecules, radionuclides, or other environmental contaminantspresent either in air, water, soil samples and/or fluid and/or tissuesamples of organisms can be assessed. An embodiment of this invention,minicells expressing proteins or other molecules could be used invariety of diagnostic detection platforms, including microwell formats,lateral flow devices, molecular switches, biosensors, badges and othersensing devices. Without being limited to the following examples, suchdevices could be used for early warning of chemical and/or bioweaponattack, illegal drug detection, explosives detection, biohazarddetection, pollution assessment, pesticide contamination, allergendetection and detection of toxic or hazardous gasses. In a relatedapplication, minicells could be used to eliminate, modify or inactivatethe agents.

[0929] In one non-limiting example of protein expression on minicellsfor toxicological detection, olfactory receptors could be expressed byminicells. The olfactory system possesses the ability to recognize anddifferentiate between a wide range of odorants based on odor moleculesinteracting with specific receptor proteins in the ciliary membrane ofolfactory neurons (Lancet, D., 1986. Vertebrate olfactory reception.Ann. Rev. Neurosci. 9:329-355; Shepherd, G. M., 1994. Discrimination ofmolecular signals by the olfactory receptor neuron. Neuron 13:771-790).These receptors were found to be 7-transmembrane-domain members of the Gprotein-coupled receptor family (Buck, L. and R. Alex. 1991. A novelmultigene family may encode odorant receptors: A molecular basis forodor recognition. Cell 65:175-187). Using a murine receptor library,olfactory receptors were functionally expressed in HEK-293 cells(Krautwurst, D., et al., 1998. Identification of ligands for olfactoryreceptors by functional expression of a receptor library. Cell.95:917-926). By coexpressing the cloned receptors with G 15,16 subunits,the modified receptor system upon activation leads to an increase inintracellular Ca²⁺. Calcium levels were measured employing the dyeFURA-2 and ratiofluormetric imaging. This system demonstrated ligandspecificity and structure-function relationships for identifiedolfactory receptors. Employing similar techniques, OR17-40, a humanolfactory receptor protein, was expressed in human embryonic kidney 293cells and Xenopus Laevis oocytes (Wetzel, H., et al. 1999. Specificityand sensitivity of a human olfactory receptor functionally expressed inHuman Embryonic Kidney 293 Cells and Xenopus Laevis Oocytes. J.Neurosciences. 19:7426-7433). The receptor was functionally expressed ina manner designed to assess the specificity of its binding to theligand, helional.

[0930] In one non-limiting example of target protein identification,primers from homologous areas in transmembrane II and transmembrane VIIof olfactory GPCRs will be used to identify unique receptor sequences.These sequences are inserted into expression vectors. Minicell producingbacteria are transformed with these vectors and cultured. Minicells areisolated from the culture as previously described and subsequentlyinduced. Using HTS previously described, the functionalreceptor/minicells which generate signal for binding of an odiferoustoxin to the receptor are identified. Large scaleLarge-scale productionof the minicells is carried out and the minicells are covalently coupledto the surface of a microarraymicro array chip. The chip is supported inan air sampler, which feeds atmosphere over surface of the chip on acontinuous basis. If the toxic agent is present in the air, the bindingto the receptor activates a series of events ending in the generation ofa signal identifying the presence of the agent in the air.

[0931] By way of non-limiting example, standard molecular biologicaltechniques can be used as follows: cDNA for GFP is ligated to the 3′ endof cDNA sequence for the receptor described above. The resultingsequence is inserted into an expression vector. Minicell producingbacteria are transformed with these vectors and cultured. Minicells areisolated from the culture as previously described and subsequentlyinduced. The minicells now contain the receptor to the ligand on thesurface of the minicell with a GFP tag on the C-terminus of the proteinin the cytosol. These minicells are packed into filters. Air is passedthrough the filter. If the ligand is present, it will bind to thereceptor. The filter packing is suspended on applied to a diagnosticdevice. Antibody to the ligand/receptor binding site complex is fixed onthe capture zone. When the sample is applied to the device, thereceptor/ligand complex is captured. The capture zone is screened forsignal resulting from the presence of GFP. This can be extrapolated tohave multiple unique receptor/minicell moieties in the same samplingdevice. Each receptor would have a unique fluorescing protein tag suchthat different emissions identify specific agents in the air.

[0932] Other methods for quantification associated take advantage of thecomposition of the minicell. Loading of the minicell, by transientlypermeabilizing the membrane to allow for migration of molecules into tothe cytosol. These molecules include but are not limited to radiolabeledmolecules ( i.e., nucleotides), stains or dyes (DAPI or other DNAstaining, heavy metals, fluorophores. The molecules could also besynthesized within the minicell (i.e. GFP). The association of aspecific ligand with the minicell could cause a redox shift that inducea color change in the solution or could shift the energy potential inthe reaction are generating an electrical current. Each of this examplesare associated with well know methods for measuring each of theresulting changes. These include but are not limited to radioactivity orfluorescence generated or the color shift by spectrophotometry.

[0933] A multigene family of gustatory G protein-coupled receptorsexpressed in the lingual epithelia has been identified with structuralsimilarities to olfactory receptors (Abe, K., et al. 1993. Multiplegenes for G protein-coupled receptors and their expression in lingualepithelia. FEBS. 316:253-256; Abe, K., et al. 1993. Primary structureand cell-type specific expression of a gustatory G protein-coupledreceptor related to olfactory receptors. J. Bio. Chem.). This providesan addition example of receptors which can be isolated, expressed inminicells and then be used for identification of specific substances invarious matrices in similar manners as identified for olfactory receptorminicells.

[0934] As a non-limiting example of minicell use intoxicological/environmental detection, arrays could be constructed inwhich each well contains a distinct minicell subtype displayingmembrane-bound proteins or other molecules for each of several potentialtoxins or agents in the environment. For example, minicells in such aformat could be used to determine which agents are present in theenvironment as a consequence of a chemical and/or biological weaponsattack. Non-limiting examples of biosensors that have been usedtoxicological/environmental detection include those described by Sticheret al., Development and characterization of a whole-cell bioluminescentsensor for bioavailable middle-chain alkanes in contaminated groundwatersamples, Appl. Envir. Microbiol. 63:4053-4060, 1997; Willardson et al.,Development and Testing of a Bacterial Biosensor for Toluene-BasedEnvironmental Contaminants, Appl. Envir. Microbiol. 64:1006-1012, 1998;Lars et al., Detection of Oxytetracycline Production by Streptomycesrimosus in Soil Microcosms by Combining Whole-Cell Biosensors and FlowCytometry, Appl. Envir. Microbiol. 67:239-244, 2001; Hø/jberg et al.,Oxygen-Sensing Reporter Strain of Pseudomonas fluorescens for Monitoringthe Distribution of Low-Oxygen Habitats in Soil, Appl. Envir. Microbiol.1999 65: 4085-4093, 1999; R. P. Hollis et al., Design and Application ofa Biosensor for Monitoring Toxicity of Compounds to Eukaryotes, Appl.Envir. Microbiol. 66: 1676-1679, 2000; Heitzer et al., Optical biosensorfor environmental on-line monitoring of naphthalene and salicylatebioavailability with an immobilized bioluminescent catabolic reporterbacterium, Appl. Envir. Microbiol. 60:1487-1494, 1994; Selifonova etal., Bioluminescent sensors for detection of bioavailable Hg(II) in theenvironment, Appl. Envir. Microbiol. 59: 3083-3090, 1993; Jaeger et al.,Mapping of Sugar and Amino Acid Availability in Soil around Roots withBacterial Sensors of Sucrose and Tryptophan, Appl. Envir. Microbiol. 65:26852690, 1999; and Larsen et al., A Microsensor for Nitrate Based onImmobilized Denitrifying Bacteria, Appl. Envir. Microbiol. 62:1248-1251, 1996.

[0935] XVIII.C. Toxin Elimination

[0936] In another embodiment of the invention, minicells displaying areceptor for a particular toxic agent could be used for the eliminationof the agent from the environment. In a non-limiting example of thistechnology, minicells could be placed in a filtering apparatus toeliminate the toxic agent from the environment (e.g., air, water soil).In the example of atmospheric contamination, the air would be circulatedthrough a forced air system containing in-line filters composed of ahousing, support matrix and receptor/minicells. As air passes over theminicells, the toxin is bound to the receptor. The purified air passedout of the system and into the atmosphere. A similar method for waterpurification would follow a similar protocol replacing the receptor forthe toxin with the receptor or other protein binding a unique epitope oncontaminant wishing to be removed. Examples include but are not limitedto removing toxins, parasites or microbes from the matrix such as wateror air. This represent non-limiting example of minicell-based technologyfor expression of functional receptors or binding moieties of receptorson the minicell's surface for the specific purpose of selectivelycapturing, identifying, quantifying and/or removing molecules ofinterest for environmental compartments to include but not limited toair water, soil, other gas phases or liquid solutions.

[0937] Representative toxins include, but are not limited to, thoseassociated with “red tides”; eubacterial toxins, such as those toxinsproduced by Corynebacterium diphtheriae (diphtheria), Bordetellapertussis (whooping cough), Vibrio cholerae (cholera), Bacillusanthracis (anthrax), Clostridium botulinum (botulism), Clostridiumtetani (tetanus), and enterohemorrhagic Escherichia coli (bloodydiarrhea and hemolytic uremic syndrome); and fungal toxins (e.g.,aflatoxin, gliotoxin, cyclopeptides, orellanine, gyrometrin, coprine,muscarine, ibotenic acid, psilocybin, psilocin and baeocystin).

[0938] The treatment of “red tides” with minicells exemplifies thisaspect of the invention. A red tide occurs as a result of ahigher-than-normal concentration of an algae or dinoflagellate which,when present in dense concentrations as a result of a “bloom,” formcolored patches on the surface of water. The colored patches are pink,violet, orange, yellow, blue, green, brown, or red, with red being themost common color. The organisms that cause red tides often producetoxins that have negative impacts on other organisms, including humans.

[0939] For example, Karenia brevis (formerly Gymnodinium breve) producesa toxin (domoic acid) that affects the central nervous system of fish,shellfish and other organisms, resulting in a state of paralysis.Alexandrium species (e.g., A. tamarense, A. fundyense), Dinophysis andGonyaulax species; and Pseudo-nitzschia multiseries, which cause,respectively, paralytic, diarrhetic and amnesic shellfish poisoning.Because shellfish containing the toxin taste and appear the same asshellfish that do not, and cooking does not destroy the toxin, humaningestion of the former can cause disease in humans and other organisms.For example, one form of paralytic shellfish poisoning, which can befatal to humans, results from saxitoxin, which is produced by Gonyaulaxtamarenis, Protogonyaulax catanella, and other species. Other algae thatcan result in red tides include Gonyaulax catenella, and Ptychdiscusbreve.

[0940] Minicells that comprise a binding moiety of an organism thatproduces a red tide, or of the toxin produced thereby, can be used forremediation. For example, a minicell having a binding moiety directed toa red tide-producing organism can be used to deliver an antibioticthereto, and a minicell with a binding moiety directed to a toxin can beused to bind and/or internalize the toxin. As is explained in moredetail elsewhere herein, a minicell with a binding moiety directed to atoxin can also be used for therapeutic purposes.

[0941] XVIII.D. Bioremediation

[0942] In another non-limiting example of the potential use of minicellsin a toxicological context is their use in bioremediation, the processby which living organisms act to degrade or transform hazardous organiccontaminants. As used herein, “bioremediation” is the process of usingbiological or biologically derived compositions that alter the chemicalstructure and/or bind, an undesirable substance in order to reduce theeffective concentration of the undesirable substance, thereby reducingor eliminating the effect(s) of the undesirable compound on theenvironment. Undesirable substances include, but are not limited to,pollutants (e.g., heavy metals, pesticides, herbicides, petroleumproducts); biological toxins (e.g., such as those produced by “redtides”, e.g., domoic acid, saxitoxin); pathogens (e.g., viruses,eubacteria); organisms that produce toxins; biological waste products(e.g., sewage, guano), and undesirable organisms therewithin (e.g.,pathogenic eubacteria).

[0943] The term “bioremediation” encompasess both biodegradation, thebreakdown of organic substances by microorganisms, andbiotransformation, the alteration of the structure of a compound by aliving organism or enzyme. The minicells of the invention may beincorporated into biofilters, i.e., devices in which gases, liquids,powders and the like are passed through media containing biodegradingminicells, including but not limited to devices that biodegrade volatileorganic compounds in air by passing the air therethrough.

[0944] Bioremedation can be used to process undesirable substances in acomposition prior to or after the release of the composition into theenvironment. For example, bioremediation can be applied in sewagetreatment plants to process sewage prior to its release, or to sewagethat has been accidentally or otherwise released into the environment.

[0945] Environmental microbiologists have sought to identify and usespecific bacteria that degrade pollutants and other environmentalcontainments. See, for example, Chakrabarty, Microbial Degradation ofToxic Chemicals: Evolutionary Insights and Practical Considerations, Am.Soc. Micro. Biol. News 62:130-137, 1996; and U.S. Pat. Nos. 4,511,657;4,493,895; 4,871,673; and 4,535,061. In instances where a live organismis placed into the environment to process undesirable substances, thereis a concern that the organism might have undesirable effects that wouldbe made more deleterious due to the ability of the live organism toreplicate (Sayler G S, Ripp S. Field applications of geneticallyengineered microorganisms for bioremediation processes. Curr OpinBiotechnol. June 2000; 11(3):286-9; and Diaz E, Ferrandez A, Prieto M A,Garcia J L. Biodegradation of aromatic compounds by Escherichia coli.Microbiol Mol Biol Rev. December 2001;65(4):523-69). For example, whenit has been proposed to use genetically altered eubacteria to processoil spills, the concern has been raised that the eubacteria might spreadbeyond the oil spill and into supplies of petroleum products that areused to produce energy, where they would process and render useless thestored petroleum products. However, because they lack the ability toreplicate, such a scenario will not occur when minicells are use forbioremediation.

[0946] By way of non-limiting example, octane enhances such as methylt-butyl ether or aromatic hydrocarbons contaminate the aquifer and soil.These agents negatively impact the many microbes in the effected areathus limiting capability of the microbial community rectify theenvironmental insult. Bioaugmentation, the addition to the environmentof microorganisms that can metabolize and grow on specific organiccompounds, to facilitate degradation may porove useful, but concernsexist relative to the regulation of newly introduced bacteria. Theminicell provides a vehicle to accomplish biodegradation withoutbacterial overgrowth.

[0947] Diphenyl ethers and cyclic ethers such as dioxane and furan haveshown to be metabolized by soil bacteria. Using classic isolation andscreening techniques identified above, genes encoding for the oxygenasesor hydroylases are isolated. The enzyme sequence is inserted into anexpression vector using standard molecular biology techniques. Minicellproducing bacteria are transformed with these vectors and cultured.Minicells are isolated from the culture as previously described andsubsequently induced. The minicells are applied to the area contaminatedwith aromatic hydrocarbons. These compounds are transported eitheractively or passively in to the minicell and subsequently degraded bythe oxygenase or hydroylase. One advantage of this focused degradationis the minimizing of feedback inhibition because the only machinery ofconsequence in the minicell is that related to the degradation of theether compounds.

[0948] Similarly, beginning with genetic material from Dehalobacterenzymes responsible for the biodegrading of tetrachloroethane could beisolated as described above. The sequence for the enzyme is insertedinto the expression vector and used to transform minicell-producingbacteria. The bacteria are cultured, minicells isolated from the cultureand the minicells induced as previously described. Minicell preps arelyophilized using standard lyophilization techniques. The resultingmaterial is transported to the site of tetrachloroethene contaminationand reconstituted and applied. As the tetracholoethene was assimilated,it is be degraded by the enzyme system.

[0949] These are non-limiting examples scope ofbioremediation/biotranformation using minicell technology. The scope ofthe invention includes taking advantage of metabolic pathways organismin general to include but not limited to eukaryotes, prokaryotes, fungi,animals or plants.

[0950] XVIII.E. Fermentation

[0951] Delivery of specific enzymes in an untargeted fashion by theminicell allows for packaged delivery without the increased biomass andcomplex metabolic products associated with processes using liveorganisms. This aspect can be taken advantage of in fermentation, wherethe addition of minicells into which unique enzymes have been added areused to modulate the composition of the environment to include but notlimited to the alcohol, sugar and acid levels.

[0952] XVIII.F. Pesticides

[0953]Bacillus thurigenesis produces a toxin that kills plant chewinginsect larvae as well as mosquito larvae. The toxin, Cry1Ac, binds toaminopeptidase N receptor on the endothelium of the midgut. Minicelltechnology is allows for delivery of the toxin. The toxin sequence ismodified by ligation of a sequence coding for a transmembrane domain aspreviously described. The sequence for this fusion protein inserted intoan expression vector using standard molecular biology techniques. Tofacilitate the consumption of the toxin/minicell plasmids containingsequences incorporating the sequence for pheromones coupled at theC-terminus to the sequence for a transmembrane domain is generated usingstandard molecular biological techniques. This fusion protein sequenceis inserted into the expression containing coding region for the toxinfusion protein or inserted into a unique expression vector. Minicellproducing bacteria are transformed with these vectors and cultured.Minicells are isolated from the culture as previously described andsubsequently induced. The minicells are distributed (e.g crop dusting)to the area of infestation. The toxin/minicells are ingested by thelarvae and kill the larvae as the minicells passes through the gut.

XIX. Pharmaceutical Compositions

[0954] Another aspect of the invention is drawn to compositions,including but not limited to pharmaceutical compositions. According tothe invention, a “composition” refers to a mixture comprising at leastone carrier, preferably a physiologically acceptable carrier, and one ormore minicell compositions. The term “carrier” defines a chemicalcompound that does not inhibit or prevent the incorporation of thebiologically active peptide(s) into cells or tissues. A carriertypically is an inert substance that allows an active ingredient to beformulated or compounded into a suitable dosage form (e.g., a pill, acapsule, a gel, a film, a tablet, a microparticle (e.g., a microsphere),a solution; an ointment; a paste, an aerosol, a droplet, a colloid or anemulsion etc.). A “physiologically acceptable carrier” is a carriersuitable for use under physiological conditions that does not abrogate(reduce, inhibit, or prevent) the biological activity and properties ofthe compound. For example, dimethyl sulfoxide (DMSO) is a carrier as itfacilitates the uptake of many organic compounds into the cells ortissues of an organism. Preferably, the carrier is a physiologicallyacceptable carrier, preferably a pharmaceutically or veterinarilyacceptable carrier, in which the minicell composition is disposed.

[0955] A “pharmaceutical composition” refers to a composition whereinthe carrier is a pharmaceutically acceptable carrier, while a“veterinary composition” is one wherein the carrier is a veterinarilyacceptable carrier. The term “pharmaceutically acceptable carrier” or“veterinarily acceptable carrier” includes any medium or material thatis not biologically or otherwise undesirable, i.e., the carrier may beadministered to an organism along with a minicell composition withoutcausing any undesirable biological effects or interacting in adeleterious manner with the complex or any of its components or theorganism. Examples of pharmaceutically acceptable reagents are providedin The United States Pharmacopeia, The National Formulary, United StatesPharmacopeial Convention, Inc., Rockville, Md. 1990, hereby incorporatedby reference herein into the present application. The terms“therapeutically effective amount” or “pharmaceutically effectiveamount” mean an amount sufficient to induce or effectuate a measurableresponse in the target cell, tissue, or body of an organism. Whatconstitutes a therapeutically effective amount will depend on a varietyof factors, which the knowledgeable practitioner will take into accountin arriving at the desired dosage regimen.

[0956] The compositions of the invention can further comprise otherchemical components, such as diluents and excipients. A “diluent” is achemical compound diluted in a solvent, preferably an aqueous solvent,that facilitates dissolution of the composition in the solvent, and itmay also serve to stabilize the biologically active form of thecomposition or one or more of its components. Salts dissolved inbuffered solutions are utilized as diluents in the art. For example,preferred diluents are buffered solutions containing one or moredifferent salts. A preferred buffered solution is phosphate bufferedsaline (particularly in conjunction with compositions intended forpharmaceutical administration), as it mimics the salt conditions ofhuman blood. Since buffer salts can control the pH of a solution at lowconcentrations, a buffered diluent rarely modifies the biologicalactivity of a biologically active peptide.

[0957] An “excipient” is any more or less inert substance that can beadded to a composition in order to confer a suitable property, forexample, a suitable consistency or to form a drug. Suitable excipientsand carriers include, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol cellulose preparations such as,for example, maize starch, wheat starch, rice starch, agar, pectin,xanthan gum, guar gum, locust bean gum, hyaluronic acid, casein potatostarch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, polyacrylate, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents can also be included, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate. Other suitable excipients and carriers includehydrogels, gellable hydrocolloids, and chitosan. Chitosan microspheresand microcapsules can be used as carriers. See WO 98/52547 (whichdescribes microsphere formulations for targeting compounds to thestomach, the formulations comprising an inner core (optionally includinga gelled hydrocolloid) containing one or more active ingredients, amembrane comprised of a water insoluble polymer (e.g., ethylcellulose)to control the release rate of the active ingredient(s), and an outerlayer comprised of a bioadhesive cationic polymer, for example, acationic polysaccharide, a cationic protein, and/or a synthetic cationicpolymer; U.S. Pat. No. 4,895,724. Typically, chitosan is cross-linkedusing a suitable agent, for example, glutaraldehyde, glyoxal,epichlorohydrin, and succinaldehyde. Compositions employing chitosan asa carrier can be formulated into a variety of dosage forms, includingpills, tablets, microparticles, and microspheres, including thoseproviding for controlled release of the active ingredient(s). Othersuitable bioadhesive cationic polymers include acidic gelatin,polygalactosamine, polyamino acids such as polylysine, polyhistidine,polyomithine, polyquaternary compounds, prolamine, polyimine,diethylaminoethyldextran (DEAE), DEAE-imine, DEAE-methacrylate,DEAE-acrylamide, DEAE-dextran, DEAE-cellulose, poly-p-aminostyrene,polyoxethane, copolymethacrylates, polyamidoamines, cationic starches,polyvinylpyridine, and polythiodiethylaminomethylethylene.

[0958] The compositions of the invention can be formulated in anysuitable manner. Minicell compositions may be uniformly (homogeneously)or non-uniformly (heterogenously) dispersed in the carrier. Suitableformulations include dry and liquid formulations. Dry formulationsinclude freeze dried and lyophilized powders, which are particularlywell suited for aerosol delivery to the sinuses or lung, or for longterm storage followed by reconstitution in a suitable diluent prior toadministration. Other preferred dry formulations include those wherein acomposition according to the invention is compressed into tablet or pillform suitable for oral administration or compounded into a sustainedrelease formulation. When the composition is intended for oraladministration but is to be delivered to epithelium in the intestines,it is preferred that the formulation be encapsulated with an entericcoating to protect the formulation and prevent premature release of theminicell compositions included therein. As those in the art willappreciate, the compositions of the invention can be placed into anysuitable dosage form. Pills and tablets represent some of such dosageforms. The compositions can also be encapsulated into any suitablecapsule or other coating material, for example, by compression, dipping,pan coating, spray drying, etc. Suitable capsules include those madefrom gelatin and starch. In turn, such capsules can be coated with oneor more additional materials, for example, and enteric coating, ifdesired. Liquid formulations include aqueous formulations, gels, andemulsions.

[0959] Some preferred embodiments concern compositions that comprise abioadhesive, preferably a mucoadhesive, coating. A “bioadhesive coating”is a coating that allows a substance (e.g., a minicellcomposition) toadhere to a biological surface or substance better than occurs absentthe coating. A “mucoadhesive coating” is a preferred bioadhesive coatingthat allows a substance, for example, a composition according to theinvention, to adhere better to mucosa occurs absent the coating. Forexample, micronized particles (e.g., particles having a mean diameter ofabout 5, 10, 25, 50, or 100 μm) can be coated with a mucoadhesive. Thecoated particles can then be assembled into a dosage form suitable fordelivery to an organism. Preferably, and depending upon the locationwhere the cell surface transport moiety to be targeted is expressed, thedosage form is then coated with another coating to protect theformulation until it reaches the desired location, where themucoadhesive enables the formulation to be retained while thecomposition interacts with the target cell surface transport moiety.

[0960] The compositions of the invention may be administered to anyorganism, preferably an animal, preferably a mammal, bird, fish, insect,or arachnid. Preferred mammals include bovine, canine, equine, feline,ovine, and porcine animals, and non-human primates. Humans areparticularly preferred. Multiple techniques of administering ordelivering a compound exist in the art including, but not limited to,oral, rectal (e.g. an enema or suppository) aerosol (e.g., for nasal orpulmonary delivery), parenteral, and topical administration. Preferably,sufficient quantities of the biologically active peptide are deliveredto achieve the intended effect. The particular amount of composition tobe delivered will depend on many factors, including the effect to beachieved, the type of organism to which the composition is delivered,delivery route, dosage regimen, and the age, health, and sex of theorganism. As such, the particular dosage of a composition incorporatedinto a given formulation is left to the ordinarily skilled artisan'sdiscretion.

[0961] Those skilled in the art will appreciate that when thecompositions of the present invention are administered as agents toachieve a particular desired biological result, which may include atherapeutic or protective effect(s) (including vaccination), it may benecessary to combine the fusion proteins of the invention with asuitable pharmaceutical carrier. The choice of pharmaceutical carrierand the preparation of the fusion protein as a therapeutic or protectiveagent will depend on the intended use and mode of administration.Suitable formulations and methods of administration of therapeuticagents include those for oral, pulmonary, nasal, buccal, ocular, dermal,rectal, or vaginal delivery.

[0962] Depending on the mode of delivery employed, the context-dependentfunctional entity can be delivered in a variety of pharmaceuticallyacceptable forms. For example, the context-dependent functional entitycan be delivered in the form of a solid, solution, emulsion, dispersion,micelle, liposome, and the like, incorporated into a pill, capsule,tablet, suppository, areosol, droplet, or spray. Pills, tablets,suppositories, areosols, powders, droplets, and sprays may have complex,multilayer structures and have a large range of sizes. Aerosols,powders, droplets, and sprays may range from small (1 micron) to large(200 micron) in size.

[0963] Pharmaceutical compositions of the present invention can be usedin the form of a solid, a lyophilized powder, a solution, an emulsion, adispersion, a micelle, a liposome, and the like, wherein the resultingcomposition contains one or more of the compounds of the presentinvention, as an active ingredient, in admixture with an organic orinorganic carrier or excipient suitable for enteral or parenteralapplications. The active ingredient may be compounded, for example, withthe usual non-toxic, pharmaceutically acceptable carriers for tablets,pellets, capsules, suppositories, solutions, emulsions, suspensions, andany other form suitable for use. The carriers which can be used includeglucose, lactose, mannose, gum acacia, gelatin, mannitol, starch paste,magnesium trisilicate, talc, corn starch, keratin, colloidal silica,potato starch, urea, medium chain length triglycerides, dextrans,andother carriers suitable for use in manufacturing preparations, in solid,semisolid, or liquid form. In addition auxiliary, stabilizing,thickening and coloring agents and perfumes may be used. Examples of astabilizing dry agent includes triulose, preferably at concentrations of0.1% or greater (See, e.g., U.S. Pat. No. 5,314,695). The activecompound is included in the pharmaceutical composition in an amountsufficient to produce the desired effect upon the process or conditionof diseases.

XX. Small Molecules

[0964] The term “small molecule” includes any chemical or other moietythat can act to affect biological processes. Small molecules can includeany number of therapeutic agents presently known and used, or can besmall molecules synthesized in a library of such molecules for thepurpose of screening for biological function(s). Small molecules aredistinguished from macromolecules by size. The small molecules of thisinvention usually have molecular weight less than about 5,000 daltons(Da), preferably less than about 2,500 Da, more preferably less than1,000 Da, most preferably less than about 500 Da.

[0965] Small molecules include without limitation organic compounds,peptidomimetics and conjugates thereof. As used herein, the term“organic compound” refers to any carbon-based compound other thanmacromolecules such nucleic acids and polypeptides. In addition tocarbon, organic compounds may contain calcium, chlorine, fluorine,copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and otherelements. An organic compound may be in an aromatic or aliphatic form.Non-limiting examples of organic compounds include acetones, alcohols,anilines, carbohydrates, monosaccharides, oligosaccharides,polysaccharides, amino acids, nucleosides, nucleotides, lipids,retinoids, steroids, proteoglycans, ketones, aldehydes, saturated,unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters,ethers, thiols, sulfides, cyclic compounds, heterocylcic compounds,imidizoles and phenols. An organic compound as used herein also includesnitrated organic compounds and halogenated (e.g., chlorinated) organiccompounds. Methods for preparing peptidomimetics are described below.Collections of small molecules, and small molecules identified accordingto the invention are characterized by techniques such as acceleratormass spectrometry (AMS; see Turteltaub et al., Curr Pharm Des 20006(10):991-1007, Bioanalytical applications of accelerator massspectrometry for pharmaceutical research; and Enjalbal et al., MassSpectrom Rev 2000 19(3):139-61, Mass spectrometry in combinatorialchemistry.)

[0966] Preferred small molecules are relatively easier and lessexpensively manufactured, formulated or otherwise prepared. Preferredsmall molecules are stable under a variety of storage conditions.Preferred small molecules may be placed in tight association withmacromolecules to form molecules that are biologically active and thathave improved pharmaceutical properties. Improved pharmaceuticalproperties include changes in circulation time, distribution,metabolism, modification, excretion, secretion, elimination, andstability that are favorable to the desired biological activity.Improved pharmaceutical properties include changes in the toxicologicaland efficacy characteristics of the chemical entity.

XXI. Polypeptides and Derivatives

[0967] XXI.A. Polypeptides

[0968] As used herein, the term “polypeptide” includes proteins, fusionproteins, oligopeptides and polypeptide derivatives, with the exceptionthat peptidomimetics are considered to be small molecules herein.Although they are polypeptides, antibodies and their derivatives aredescribed in a separate section. Antibodies and antibody derivatives aredescribed in a separate section, but antibodies and antibody derivativesare, for purposes of the invention, treated as a subclass of thepolypeptides and derivatives.

[0969] A “protein” is a molecule having a sequence of amino acids thatare linked to each other in a linear molecule by peptide bonds. The termprotein refers to a polypeptide that is isolated from a natural source,or produced from an isolated cDNA using recombinant DNA technology; andhas a sequence of amino acids having a length of at least about 200amino acids.

[0970] A “fusion protein” is a type of recombinant protein that has anamino acid sequence that results from the linkage of the amino acidsequences of two or more normally separate polypeptides.

[0971] A “protein fragment” is a proteolytic fragment of a largerpolypeptide, which may be a protein or a fusion protein. A proteolyticfragment may be prepared by in vivo or in vitro proteolytic cleavage ofa larger polypeptide, and is generally too large to be prepared bychemical synthesis. Proteolytic fragments have amino acid sequenceshaving a length from about 200 to about 1,000 amino acids.

[0972] An “oligopeptide” is a polypeptide having a short amino acidsequence (i.e., 2 to about 200 amino acids). An oligopeptide isgenerally prepared by chemical synthesis.

[0973] Although oligopeptides and protein fragments may be otherwiseprepared, it is possible to use recombinant DNA technology and/or invitro biochemical manipulations. For example, a nucleic acid encoding anamino acid sequence may be prepared and used as a template for in vitrotranscription/translation reactions. In such reactions, an exogenousnucleic acid encoding a preselected polypeptide is introduced into amixture that is essentially depleted of exogenous nucleic acids thatcontains all of the cellular components required for transcription andtranslation. One or more radiolabeled amino acids are added before orwith the exogenous DNA, and transcription and translation are allowed toproceed. Because the only nucleic acid present in the reaction mix isthe exogenous nucleic acid added to the reaction, only polypeptidesencoded thereby are produced, and incorporate the radiolabelled aminoacid(s). In this manner, polypeptides encoded by a preselected exogenousnucleic acid are radiolabeled. Although other proteins are present inthe reaction mix, the preselected polypeptide is the only one that isproduced in the presence of the radiolabeled amino acids and is thusuniquely labeled.

[0974] As is explained in detail below, “polypeptide derivatives”include without limitation mutant polypeptides, chemically modifiedpolypeptides, and peptidomimetics.

[0975] The polypeptides of this invention, including the analogs andother modified variants, may generally be prepared following knowntechniques. Preferably, synthetic production of the polypeptide of theinvention may be according to the solid phase synthetic method. Forexample, the solid phase synthesis is well understood and is a commonmethod for preparation of polypeptides, as are a variety ofmodifications of that technique [Merrifield (1964), J. Am. Chem. Soc.,85: 2149; Stewart and Young (1984), Solid Phase polypeptide Synthesis,Pierce Chemical Company, Rockford, Ill.; Bodansky and Bodanszky (1984),The Practice of polypeptide Synthesis, Springer-Verlag, New York;Atherton and Sheppard (1989), Solid Phase polypeptide Synthesis: APractical Approach, IRL Press, New York]. See, also, the specific methoddescribed in Example 1 below.

[0976] Alternatively, polypeptides of this invention may be prepared inrecombinant systems using polynucleotide sequences encoding thepolypeptides. For example, fusion proteins are typically prepared usingrecombinant DNA technology.

[0977] XXI.B. Polypeptide Derivatives

[0978] A “derivative” of a polypeptide is a compound that is not, bydefinition, a polypeptide, i.e., it contains at least one chemicallinkage that is not a peptide bond. Thus, polypeptide derivativesinclude without limitation proteins that naturally undergopost-translational modifications such as, e.g., glycosylation. It isunderstood that a polypeptide of the invention may contain more than oneof the following modifications within the same polypeptide. Preferredpolypeptide derivatives retain a desirable attribute, which may bebiological activity; more preferably, a polypeptide derivative isenhanced with regard to one or more desirable attributes, or has one ormore desirable attributes not found in the parent polypeptide. Althoughthey are described in this section, peptidomimetics are taken as smallmolecules in the present disclosure.

[0979] XXI.C. Mutant Polypeptide Derivatives

[0980] A polypeptide having an amino acid sequence identical to thatfound in a protein prepared from a natural source is a “wildtype”polypeptide. Mutant oligopeptides can be prepared by chemical synthesis,including without limitation combinatorial synthesis.

[0981] Mutant polypeptides larger than oligopeptides can be preparedusing recombinant DNA technology by altering the nucleotide sequence ofa nucleic acid encoding a polypeptide. Although some alterations in thenucleotide sequence will not alter the amino acid sequence of thepolypeptide encoded thereby (“silent” mutations), many will result in apolypeptide having an altered amino acid sequence that is alteredrelative to the parent sequence. Such altered amino acid sequences maycomprise substitutions, deletions and additions of amino acids, with theproviso that such amino acids are naturally occurring amino acids.

[0982] Thus, subjecting a nucleic acid that encodes a polypeptide tomutagenesis is one technique that can be used to prepare mutantpolypeptides, particularly ones having substitutions of amino acids butno deletions or insertions thereof. A variety of mutagenic techniquesare known that can be used in vitro or in vivo including withoutlimitation chemical mutagenesis and PCR-mediated mutagenesis. Suchmutagenesis may be randomly targeted (i.e., mutations may occur anywherewithin the nucleic acid) or directed to a section of the nucleic acidthat encodes a stretch of amino acids of particular interest. Using suchtechniques, it is possible to prepare randomized, combinatorial orfocused compound libraries, pools and mixtures.

[0983] Polypeptides having deletions or insertions of naturallyoccurring amino acids may be synthetic oligopeptides that result fromthe chemical synthesis of amino acid sequences that are based on theamino acid sequence of a parent polypeptide but which have one or moreamino acids inserted or deleted relative to the sequence of the parentpolypeptide. Insertions and deletions of amino acid residues inpolypeptides having longer amino acid sequences may be prepared bydirected mutagenesis.

[0984] XXI.D. Chemically Modified Polypeptides

[0985] As contemplated by this invention, the term “polypeptide”includes those having one or more chemical modification relative toanother polypeptide, i.e., chemically modified polypeptides. Thepolypeptide from which a chemically modified polypeptide is derived maybe a wildtype protein, a mutant protein or a mutant polypeptide, orpolypeptide fragments thereof; an antibody or other polypeptide ligandaccording to the invention including without limitation single-chainantibodies, bacterial proteins and polypeptide derivatives thereof; orpolypeptide ligands prepared according to the disclosure. Preferably,the chemical modification(s) confer(s) or improve(s) desirableattributes of the polypeptide but does not substantially alter orcompromise the biological activity thereof. Desirable attributes includebut are limited to increased shelf-life; enhanced serum or other in vivostability; resistance to proteases; and the like. Such modificationsinclude by way of non-limiting example N-terminal acetylation,glycosylation, and biotinylation.

[0986] XXI.D.1. Polypeptides with N-Terminal or C-Terminal ChemicalGroups

[0987] An effective approach to confer resistance to peptidases actingon the N-terminal or C-terminal residues of a polypeptide is to addchemical groups at the polypeptide termini, such that the modifiedpolypeptide is no longer a substrate for the peptidase. One suchchemical modification is glycosylation of the polypeptides at either orboth termini. Certain chemical modifications, in particular N-terminalglycosylation, have been shown to increase the stability of polypeptidesin human serum (Powell et al. (1993), Pharma. Res. 10: 1268-1273). Otherchemical modifications which enhance serum stability include, but arenot limited to, the addition of an N-terminal alkyl group, consisting ofa lower alkyl of from 1 to 20 carbons, such as an acetyl group, and/orthe addition of a C-terminal amide or substituted amide group.

[0988] XXI.D.2. Polypeptides with a Terminal D-Amino Acid

[0989] The presence of an N-terminal D-amino acid increases the serumstability of a polypeptide that otherwise contains L-amino acids,because exopeptidases acting on the N-terminal residue cannot utilize aD-amino acid as a substrate. Similarly, the presence of a C-terminalD-amino acid also stabilizes a polypeptide, because serum exopeptidasesacting on the C-terminal residue cannot utilize a D-amino acid as asubstrate. With the exception of these terminal modifications, the aminoacid sequences of polypeptides with N-terminal and/or C-terminal D-aminoacids are usually identical to the sequences of the parent L-amino acidpolypeptide.

[0990] XXI.D.3. Polypeptides With Substitution of Natural Amino Acids ByUnnatural Amino Acids

[0991] Substitution of unnatural amino acids for natural amino acids ina subsequence of a polypeptide can confer or enhance desirableattributes including biological activity. Such a substitution can, forexample, confer resistance to proteolysis by exopeptidases acting on theN-terminus. The synthesis of polypeptides with unnatural amino acids isroutine and known in the art (see, for example, Coller, et al. (1993),cited above).

[0992] XXI.D.4. Post-Translational Chemical Modifications

[0993] Different host cells will contain different post-translationalmodification mechanisms that may provide particular types ofpost-translational modification of a fusion protein if the amino acidsequences required for such modifications is present in the fusionprotein. A large number (˜100) of post-translational modifications havebeen described, a few of which are discussed herein. One skilled in theart will be able to choose appropriate host cells, and design chimericgenes that encode protein members comprising the amino acid sequenceneeded for a particular type of modification.

[0994] Glycosylation is one type of post-translational chemicalmodification that occurs in many eukaryotic systems, and may influencethe activity, stability, pharmacogenetics, immunogenicity and/orantigenicity of proteins. However, specific amino acids must be presentat such sites to recruit the appropriate glycosylation machinery, andnot all host cells have the appropriate molecular machinery.Saccharomyces cerevisieae and Pichia pastoris provide for the productionof glycosylated proteins, as do expression systems that utilize insectcells, although the pattern of glyscoylation may vary depending on whichhost cells are used to produce the fusion protein.

[0995] Another type of post-translation modification is thephosphorylation of a free hydroxyl group of the side chain of one ormore Ser, Thr or Tyr residues. Protein kinases catalyze such reactions.Phosphorylation is often reversible due to the action of a proteinphosphatase, an enzyme that catalyzes the dephosphorylation of aminoacid residues.

[0996] Differences in the chemical structure of amino terminal residuesresult from different host cells, each of which may have a differentchemical version of the methionine residue encoded by a start codon, andthese will result in amino termini with different chemicalmodifications.

[0997] For example, many or most bacterial proteins are synthesized withan amino terminal amino acid that is a modified form of methionine, i.e,N-formyl-methionine (fMet). Although the statement is often made thatall bacterial proteins are synthesized with an fMet initiator aminoacid; although this may be true for E. coli, recent studies have shownthat it is not true in the case of other bacteria such as Pseudomonasaeruginosa (Newton et al., J. Biol. Chem. 274:22143-22146, 1999). In anyevent, in E. coli, the formyl group of fMet is usually enzymaticallyremoved after translation to yield an amino terminal methionine residue,although the entire fMet residue is sometimes removed (see Hershey,Chapter 40, “Protein Synthesis” in: Escherichia Coli and SalmonellaTyphimurium: Cellular and Molecular Biology, Neidhardt, Frederick C.,Editor in Chief, American Society for Microbiology, Washington, D.C.,1987, Volume 1, pages 613-647, and references cited therein.) E. colimutants that lack the enzymes (such as, e.g., formylase) that catalyzesuch post-translational modifications will produce proteins having anamino terminal fMet residue (Guillon et al., J. Bacteriol.174:4294-4301, 1992).

[0998] In eukaryotes, acetylation of the initiator methionine residue,or the penultimate residue if the initiator methionine has been removed,typically occurs co- or post-translationally. The acetylation reactionsare catalyzed by N-terminal acetyltransferases (NATs, a.k.a.N-alpha-acetyltransferases), whereas removal of the initiator methionineresidue is catalyzed by methionine aminopeptidases (for reviews, seeBradshaw et al., Trends Biochem. Sci. 23:263-267, 1998; and Driessen etal., CRC Crit. Rev. Biochem. 18:281-325, 1985). Amino terminallyacetylated proteins are said to be “N-acetylated,” “N alpha acetylated”or simply “acetylated.”

[0999] Another post-translational process that occurs in eukaryotes isthe alpha-amidation of the carboxy terminus. For reviews, see Eipper etal. Annu. Rev. Physiol. 50:333-344, 1988, and Bradbury et al. LungCancer 14:239-251, 1996. About 50% of known endocrine and neuroendocrinepeptide hormones are alpha-amidated (Treston et al., Cell Growth Differ.4:911-920, 1993). In most cases, carboxy alpha-amidation is required toactivate these peptide hormones.

[1000] XXI.E. Peptidomimetics

[1001] In general, a polypeptide mimetic (“peptidomimetic”) is amolecule that mimics the biological activity of a polypeptide but is nolonger peptidic in chemical nature. By strict definition, apeptidomimetic is a molecule that contains no peptide bonds (that is,amide bonds between amino acids). However, the term peptidomimetic issometimes used to describe molecules that are no longer completelypeptidic in nature, such as pseudo-peptides, semi-peptides and peptoids.Examples of some peptidomimetics by the broader definition (where partof a polypeptide is replaced by a structure lacking peptide bonds) aredescribed below. Whether completely or partially non-peptide,peptidomimetics according to this invention provide a spatialarrangement of reactive chemical moieties that closely resembles thethree-dimensional arrangement of active groups in the polypeptide onwhich the peptidomimetic is based. As a result of this similaractive-site geometry, the peptidomimetic has effects on biologicalsystems that are similar to the biological activity of the polypeptide.

[1002] There are several potential advantages for using a mimetic of agiven polypeptide rather than the polypeptide itself. For example,polypeptides may exhibit two undesirable attributes, i.e., poorbioavailability and short duration of action. Peptidomimetics are oftensmall enough to be both orally active and to have a long duration ofaction. There are also problems associated with stability, storage andimmunoreactivity for polypeptides that are not experienced withpeptidomimetics.

[1003] Candidate, lead and other polypeptides having a desiredbiological activity can be used in the development of peptidomimeticswith similar biological activities. Techniques of developingpeptidomimetics from polypeptides are known. Peptide bonds can bereplaced by non-peptide bonds that allow the peptidomimetic to adopt asimilar structure, and therefore biological activity, to the originalpolypeptide. Further modifications can also be made by replacingchemical groups of the amino acids with other chemical groups of similarstructure. The development of peptidomimetics can be aided bydetermining the tertiary structure of the original polypeptide, eitherfree or bound to a ligand, by NMR spectroscopy, crystallography and/orcomputer-aided molecular modeling. These techniques aid in thedevelopment of novel compositions of higher potency and/or greaterbioavailability and/or greater stability than the original polypeptide(Dean (1994), BioEssays, 16: 683-687; Cohen and Shatzmiller (1993), J.Mol. Graph., 11: 166-173; Wiley and Rich (1993), Med. Res. Rev., 13:327-384; Moore (1994), Trends Pharmacol. Sci., 15: 124-129; Hruby(1993), Biopolymers, 33: 1073-1082; Bugg et al. (1993), Sci. Am., 269:92-98, all incorporated herein by reference].

[1004] Thus, through use of the methods described above, the presentinvention provides compounds exhibiting enhanced therapeutic activity incomparison to the polypeptides described above. The peptidomimeticcompounds obtained by the above methods, having the biological activityof the above named polypeptides and similar three-dimensional structure,are encompassed by this invention. It will be readily apparent to oneskilled in the art that a peptidomimetic can be generated from any ofthe modified polypeptides described in the previous section or from apolypeptide bearing more than one of the modifications described fromthe previous section. It will furthermore be apparent that thepeptidomimetics of this invention can be further used for thedevelopment of even more potent non-peptidic compounds, in addition totheir utility as therapeutic compounds.

[1005] Specific examples of peptidomimetics derived from thepolypeptides described in the previous section are presented below.These examples are illustrative and not limiting in terms of the otheror additional modifications.

[1006] XXI.E. 1. Peptides With A Reduced Isostere Pseudopeptide Bond

[1007] Proteases act on peptide bonds. It therefore follows thatsubstitution of peptide bonds by pseudopeptide bonds confers resistanceto proteolysis. A number of pseudopeptide bonds have been described thatin general do not affect polypeptide structure and biological activity.The reduced isostere pseudopeptide bond is a suitable pseudopeptide bondthat is known to enhance stability to enzymatic cleavage with no orlittle loss of biological activity (Couder, et al. (1993), Int. J.Polypeptide Protein Res. 41:181-184, incorporated herein by reference).Thus, the amino acid sequences of these compounds may be identical tothe sequences of their parent L-amino acid polypeptides, except that oneor more of the peptide bonds are replaced by an isostere pseudopeptidebond. Preferably the most N-terminal peptide bond is substituted, sincesuch a substitution would confer resistance to proteolysis byexopeptidases acting on the N-terminus.

[1008] XXI.E.2. Peptides With A Retro-Inverso Pseudopeptide Bond

[1009] To confer resistance to proteolysis, peptide bonds may also besubstituted by retro-inverso pseudopeptide bonds (Dalpozzo, et al.(1993), Int. J. Polypeptide Protein Res. 41:561-566, incorporated hereinby reference). According to this modification, the amino acid sequencesof the compounds may be identical to the sequences of their L-amino acidparent polypeptides, except that one or more of the peptide bonds arereplaced by a retro-inverso pseudopeptide bond. Preferably the mostN-terminal peptide bond is substituted, since such a substitution willconfer resistance to proteolysis by exopeptidases acting on theN-terminus.

[1010] XXI.E.3. Peptoid Derivatives

[1011] Peptoid derivatives of polypeptides represent another form ofmodified polypeptides that retain the important structural determinantsfor biological activity, yet eliminate the peptide bonds, therebyconferring resistance to proteolysis (Simon, et al., 1992, Proc. Natl.Acad. Sci. USA, 89:9367-9371 and incorporated herein by reference).Peptoids are oligomers of N-substituted glycines. A number of N-alkylgroups have been described, each corresponding to the side chain of anatural amino acid.

XXII. Kits

[1012] The invention provides for diagnostic and therapeutic kitsrelated useful for therapeutic, diagnostic, and research applications.Exemplary kits are disclosed in U.S. Pat. Nos. 5,773,024; 6,017,721; and6,232,127 B1. The kits of the invention incorporate minicells, and/orinclude methods of using minicells described herein.

[1013] XXII.A. Diagnostic and Research Use Kit Components

[1014] In one embodiment, the invention relates to kits for determiningthe diagnosis or prognosis of a patient. These kits preferably comprisedevices and reagents for measuring one or more marker levels in a testsample from a patient, and instructions for performing the assay.Optionally, the kits may contain one or more means for converting markerlevel(s) to a prognosis. Such kits preferably contain sufficientreagents to perform one or more such determinations.

[1015] More specifically, a diagnostic kit of the invention comprisesany of the following reagents and/or components in any combination.

[1016] (1) A detectable or detectably labeled first reagent that binds aligand of interest. The binding reagent can, but need not, be anantibody or an antibody derivative comprising a detectable moiety. Thesphingolipid-binding reagent is stored in an openable container in thekit, or is bound to a surface of a substrate such that it is accessibleto other reagents. Examples of the latter include test strips.

[1017] (2) If the first reagent in neither detectable nor detectablylabeled, the kit may comprise a detectable or detectably labeled secondreagent that binds to the first reagent (e.g., a secondary antibody) orwhich produces a detectable signal when in close proximity to the firstreagent (e.g., as results from fluorescent resonance energy transferFRET). In either case, the signal produced from the second reagentcorrelates with the amount of ligand in the sample.

[1018] (3) One or more positive control reagents. Typically, thesereagents comprise a compound that is known to produce a signal in theassay. In one embodiment, the positive control reagents are standards,i.e., comprise a known amount of a detectable or detectably labeledcompound, the signal from which may be compared to the signal from atest sample. In addition to serving as positive control reagents, theymay be used to develop calibration curves that relate the amount ofsignal to the known concentration of a detectable or detectably labeledcompound. The signal from a test sample is compared to the calibrationcurve in order to determine what concentration of the detectable ordetectably labeled compound corresponds to the signal from the testsample. In this embodiment, the kit provides quantitative measurementsof the amount of a ligand in a test sample.

[1019] (4) One or more negative control reagents. Typically, thesecontrol reagents may comprise buffer or another solution that does notcontain any of the detectable or detectably labeled first or secondreagents and should thus not produce any detectable signal. Any signalthat is detected reflects the background level of “noise” in the assay.Another type of negative control reagent contains most of the componentsnecessary for the signal of the assay to be produced, but lacks at leastone such component and therefor should not produce a signal. Yet anothertype of negative control reagent contains all of the componentsnecessary for the signal of the assay to be produced, but also containsan inhibitor of the process that produced the signal.

[1020] (5) One or more auxiliary reagents for use in the diagnosticassays of the kit, e.g., buffers, alcohols, acid solutions, etc. Thesereagents are generally available in medical facilities and thus areoptional components of the kit. However, these reagents preferably areincluded in the kit to ensure that reagents of sufficient purity andsterility are used, since the resulting protein conjugates are to beadministered to mammals, including humans, for medical purposes, and toprovide kits that can be used in situations where medical facilities arenot readily available, e.g., when hiking in places located far frommedical facilities, or in situations where the presence of theseauxiliary reagents allows for the immediate treatment of a patientoutside of a medical facility as opposed to treatment that arrives atsome later time).

[1021] (6) Instructions to a person using a kit for its use. Theinstructions can be present on one or more of the kit components, thekit packaging and/or a kit package insert.XXII.B. Therapeutic KitComponents

[1022] A therapeutic kit of the invention comprises any of the followingreagents and/or components in any combination.

[1023] (1) One or more therapeutic agents.

[1024] (2) If the therapeutic agent(s) are not formulated for deliveryvia the alimentary canal, which includes but is not limited tosublingual delivery, a device capable of delivering the therapeuticagent through some other routes. One type of device for parenteraldelivery is a syringe that is used to inject the therapeutic agent intothe body of an animal in need of the therapeutic agent. Inhalationdevices may also be used.

[1025] (3) Separate containers, each of which comprises one or morereagents of the kit. In a preferred embodiment, the containers are vialscontain sterile, lyophilized formulations of a therapeutic compositionthat are suitable for reconstitution. Other containers include, but arenot limited to, a pouch, tray, box, tube, or the like. Kit componentsmay be packaged and maintained sterilely within the containers.

[1026] (4) Instructions to a person using a kit for its use. Theinstructions can be present on one or more of the kit components, thekit packaging and/or a kit package insert. Such instructions include, byway of non-limiting example, instructions for use of the kit and itsreagents, for reconstituting lyophilized reagents or otherwise preparingreagents.

[1027] A preferred kit of the present invention comprises the elementsuseful for performing an immunoassay. A kit of the present invention cancomprise one or more experimental samples (i.e., formulations of thepresent invention) and one or more control samples bound to at least onepre-packed dipstick or ELISA plate, and the necessary means fordetecting immunocomplex formation (e.g., labelled secondary antibodiesor other binding compounds and any necessary solutions needed to resolvesuch labels, as described in detail above) between antibodies containedin the bodily fluid of the animal being tested and the proteins bound tothe dipstick or ELISA plate. It is within the scope of the inventionthat the kit can comprise simply a formulation of the present inventionand that the detecting means can be provided in another way.

[1028] An alternative preferred kit of the present invention compriseselements useful for performing a skin test. A kit of the presentinvention can comprise at least one pre-packed syringe and needleapparatus containing one or more experimental samples and/or one or morecontrol samples. A kit according to the invention may be designed forboth diagnostic and therapeutiuc applications. Any combination of theabove elements XX.A.(1)-(6) and XX.B.(1)-(4) may be used in a kit,optionally with additional reagents, standards, sample containers, anthe like.

XXIII. Immunogenic Minicells

[1029] XXIII.A. In General

[1030] Minicells are used to immunize subjects. An organism is said tobe “immunized” when, after contact with an immunogen, the organismproduces antibodies directed to the immunogen, or has increasedproliferation or activity of cytotoxic and/or helper T cells, or both.Increased proliferation or activity of T cells may be particularlydesirable in the case of parasites that cause a decrease in T cellproliferation.

[1031] The use of minicells to present antigens has several potentialadvantages. An intact membrane protein can be presented in its nativeform on the surface of an immunogenic minicell, rather than as adenatured protein or as oligopeptides derived from the amino acidsequence of a membrane protein, which allows for antibodies to bedeveloped that are directed to epitopes which, due to protein folding,occur only in the native protein. The minicell surface may naturally be,or may be modified to be, an adjuvant. Moreover, pharmacokineticproperties of minicells, as discussed elsewhere herein, may be improvedrelative to other forms of administration.

[1032] The applications of immunogenic minicells include, but are notlimited to, research, prophylactic, diagnostic and therapeuticapplications.

[1033] In research applications, immunogenic minicells are used togenerate antibodies to an antigen displayed on a minicell. Suchantibodies are used to detect an antigen, which may be a chemicalmoiety, molecule, virus, organelle, cell, tissue, organ, or organismthat one wishes to study.

[1034] Classically, such antibodies have been prepared by immunizing ananimal, often a rat or a rabbit, and collecting antisera therefrom.Molecular biology techniques can be used to prepare antibodies andantibody fragments, as is described elsewhere herein. Single-chainantibody fragments (scFv) may also be identified, purified, andcharacterized using minicells displaying a membrane protein or membranebound chimeric soluble protein.

[1035] In prophylactic applications, immunogenic minicells are used tostimulate a subject to produce antibodies and/or activate T cells, sothat the subject is “pre-immunized” before contact with a pathogen orhyperproliferative cell. Thus, in the case of a pathogens, the subjectis protected by antibodies and/or T cells that are specifically directedto the pathogen before infection.

[1036] In therapeutic applications, immunogenic minicells are used inimmunotherapy.

[1037] Certain aspects of the invention involve active immunotherapy, inwhich treatment relies on the in vivo stimulation of the endogenous hostimmune system to react against pathogens or tumors due to theadministration of agents that cause, enhance or modulate an immuneresponse. Such agents include, but are not limited to, immunogens,adjuvants, cytokines and chemokines.

[1038] Other therapeutic applications involve passive immunotherapy, inwhich treatment involves the delivery of agents (such as antibodies oreffector cells) that are specifically directed to an immunogen of apathogen or a hyperproliferative cell, and does not necessarily dependon an intact host immune system. Examples of effector cells include Tcells; T lymphocytes, such as CD8+ cytotoxic T lymphocytes and CD4+T-helper tumor-infiltrating lymphocytes; killer cells, such as NaturalKiller (NK) cells and lymphokine-activated killer cells.

[1039] XXIII.B. Hyperproliferative Disorders

[1040] The immunogenic minicells of the invention can be used to treathyperproliferative disorders by inducing an immune response to anantigen associated therewith. The term “hyperproliferative disorder”refers to disorders characterized by an abnormal or pathologicalproliferation of cells, for example, cancer, psoriasis, hyperplasia andthe like.

[1041] For reviews of immunotherapy as applied to hyperproliferativedisorders, see Armstrong et al., Cellular immunotherapy for cancer, BMJ323:1289-1293, 2001; Evans, Vaccine therapy for cancer—fact or fiction?,Proc R Cell Physicians Edinb 31:9-16, 2001; Ravindranath and Morton,“Active Specific Immunotherapy with Vaccines,” Chapter 61 in:Holland-Frei Cancer Medicine, Fifth Edition, Bast, Robert C., et al.,editors, B.C. Decker, Inc., Hamilton, 2000, pages 800-814.

[1042] Types of cancers include without limitation fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom'smacroglobulinemia, and heavy chain disease.

[1043] Tumor specific antigens (TSAs), tumor-associated differentiationantigens (TADAs) and other antigens associated with cancers and otherhyperproliferative disorders include, but are not limited to, C1 IAC, ahuman cancer associated protein (Osther, U.S. Pat. No. 4,132,769); theCA125 antigen, an antigen associated with cystadenocarcinoma of theovary, (Hanisch et al., Carbohydr. Res. 178:29-47, 1988; O'Brien, U.S.Pat. No. 4,921,790); CEA, an antigen present on many adenocarcinomas(Horig et al., Strategies for cancer therapy using carcinembryonicantigen vaccines, Expert Reviews in Molecular Medicine,http://www-ermm.cbcu.cam.ac.uk: 1,2000); CORA (carcinoma ororosomucoid-related antigen) described by Toth et al. (U.S. Pat. No.4,914,021); DF3 antigen from human breast carcinoma (Kufe, in U.S. Pat.Nos. 4,963,484 and 5,053,489); DU-PAN-2, a pancreatic carcinoma antigen(Lan et al., Cancer Res. 45:305-310, 1985); HCA, a human carcinomaantigen (Codington et al., U.S. Pat. No. 5,693,763); Her2, a breastcancer antigen (Fendly et al., The Extracellular Domain of HER2/neu Is aPotential Immunogen for Active Specific Immunotherapy of Breast Cancer,Journal of Biological Response Modifiers 9:449-455, 1990); MSA, a breastcarcinoma glycoprotein (Tjandra et al., Br. J. Surg. 75:811-817, 1988);MFGM, a breast carcinoma antigen (Ishida et al., Tumor Biol. 10:12-22,1989); PSA, prostrate specific antigen (Nadji et al.,Prostatic-specific-antigen, Cancer 48:1229-1232, 1981); STEAP (sixtransmembrane epithelial antigens of the prostate) proteins (Afar etal., U.S. Pat. No. 6,329,503); TAG-72, a breast carcinoma glycoprotein(Kjeldsen et al., Cancer Res. 48:2214-2220, 1988); YH206, a lungcarcinoma antigen (Hinoda et al., Cancer J. 42:653-658, 1988); the p97antigen of human melanoma (Estin et al., Recombinant Vaccinia VirusVaccine Against the Human Melanoma Antigen p97 for Use in Immunotherapy,Proc. Natl Acad. Sci. USA, 85:1052-1056, 1988); and the melanomaspecific antigen described by Pfreundschuh in U.S. Pat. No. 6,025,191);

[1044] XXIII.B. Intracellular Pathogens

[1045] In certain aspects of the invention, vaccines comprisingimmunogenic minicells are used to prevent or treat diseases caused byintracellular pathogens. Vaccines may be prepared that stimulatecytotoxic T cell responses against cells infected with virusesincluding, but not limited to, hepatitis type A, hepatitis type B,hepatitis type C, influenza, varicella, adenovirus, herpes simplex typeI (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirous,echovirus, rotavirus, respiratory syncytial virus, papilloma virus,papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus,coxsackie virus, mumps virus, measles virus, rubella virus, polio virus,human immunodeficiency virus type I (HIV-I), and human immunodeficiencyvirus type II (HIV-II). Vaccines also may be prepared that stimulatecytotoxic T cell responses against cells infected with intracellularobligates, including but not limited to Chlamydia, Mycobacteria andRickettsia. Vaccines also may be prepared that stimulate cytotoxic Tcell responses against cells infected with intracellular protozoa,including, but not limited to, leishmania, kokzidioa, and trypanosoma.

[1046] The causative agent of Lyme disease, the spirochete Borreliaburgdorfei, is also of interest. The outer surface proteins (Osps) A, Band C of B. burgdorfei are known antigens that are lipoproteins thatassociate with membranes. Amino-terminal cysteine residues in Ospproteins are the sites of triacyl lipid modifications that serve asmembrane-anchoring moities. The N-terminal portions of the Osp proteinsare highly conserved and are preferred portions for display onimmunogenic minicells.

[1047] XXIII.C. Eukaryotic Pathogens

[1048] In addition to intracellular pathogens, other eukaryoticpathogens exist and may also be treated using immunogenic minicellsdisplayed antigens therefrom. A number of antigens have been used todevelop anti-parasitic vaccines, e.g. the recombinant 45w protein ofTaenia ovis; EG95 oncosphere proteins of Echinococcus granulosis;cathepsin L antigen of the liver fluke, Fasciola hepatica; and the H11antigen of Haemonchus contortus (Dalton et al., Parasite vaccines—areality?, Vet Parasitol 98:149-167, 2001). Other eukaryotic pathogensinclude, but are not limited to:

[1049] Protozoans, including but not limited to, Entamoeba histolytica,a pathogenic amoeba that causes amoebic dysentery and occasionallydigests its way through the intestinal wall to invades other organs,which may cause morbidity; Balantinium coli, a ciliate that causesdiarrhea in humans; Giardia lamblia, a flagellate that causes diarrheaand abdominal pain, along with a chronic fatigue syndrome that isotherwise asymptomatic and difficult to diagnose; Trypanosoma brucei, ahemoflagellate causing sleeping sickness; and Trypanosoma cruzi, thecause of Chagas disease);

[1050] Plasmodia, sporozoan obligate intracellular parasites of liverand red blood cells, including but not limited to P. falciparum, thecausative agent of malaria. Dozens of P. falciparum antigens have beenidentified, e.g., CSP-1, STARP, SALSA, SSP-2, LSA-1, EXP-1, LSA-3,RAP-1, RAP-2, SERA-1, MSP-1, MSP-2, MSP-3, MSP-4, MSP-5, AMA-1, EBA-175,RESA, GLURP, EMP-1, Pfs25, Pfg27, Pf35, Pf55, Pfs230, Pfg27, Pfs16,Pfs28 and Pfs45/48.

[1051] Helminthes including but not limited to Ascaris lumbricoides(roundworm); Enterobius vermicularis (pinworm); Trichuris trichiuria(whipworm); and Fasciola hepatica (liver fluke);

[1052] Taenia sp. (tapeworms and cestodes);

[1053] Schistosoma (trematodes), such as Schistoma mansoni, whichcomprises the Sm32 antigen (asparaginyl endopeptidase), which can induceantibody formation in mice (Chlichlia et al., DNA vaccination withasparaginyl endopeptidase (Sm32) from the parasite Schistosoma mansoni:anti-fecundity effect induced in mice, Vaccine 20:439-447, 2001); andacetylcholinesterase (Arnon et al., Acetylcholinesterase of Schistomamansoni-Functional correlates, Protein Science 8:2553-2561, 1999); and

[1054] Ticks and other invertebrates, including but not limited toinsects, arachnids, etc. For example, a description of a vaccine againstthe cattle tick Boophilus microplus has been described (Valle et al.,The evaluation of yeast derivatives as adjuvants for the immune responseto the Bm86 antigen in cattle, BMC Biotechnol. 1:2, 2001)

[1055] XXIII.D. Formulation and Adminstration of Immunogenic Minicells

[1056] Vaccine formulations of immunogenic minicells include a suitablecarrier. Because minicells may be destroyed by digestion, or preventedfrom acting due to antibody secretion in the gvut, they are preferablyadministered parenterally, including, for example, administration thatis subcutaneous, intramuscular, intravenous, or intradermal.Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain antioxidanits,buffers, and solutes which render the formulation isotonic with thebodily fluid, preferably the blood, of the individual; and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampules and vials and may bestored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The vaccine formulationmay also include adjuvant systems for enhancing the immunogenicity ofthe formulation. Adjuvants are substances that can be used to augment aspecific immune response. Normally, the adjuvant and the composition aremixed prior to presentation to the immune system, or presentedseparately, but into the same site of the mammal being immunized.Examples of materials suitable for use in vaccine compositions areprovided in Osol, A., ed., Remington's Pharmaceutical Sciences, MackPublishing Co, Easton, Pa. (1980), pp. 1324-1341, which reference isentirely incorporated herein by reference.

[1057] Compositions comprising immunogenic minicells are injected into ahuman or animal at a dosage of 1-1000 ug per kg body weight. Antibodytiters against growth factor are determined by ELISA, using therecombinant protein and horseradish peroxidase-conjugated goatanti-human or animal immunoglobulins or other serologic techniques(e.g., sandwich ELISA). Booster injections are administered as needed toachieve the desired levels of protective antibodies and/or T cells.

[1058] Routes and frequency of administration, as well as dosage, willvary from individual to individual. Between 1 and 10 doses may beadministered for a 52-week period. Preferably, 6 doses are administered,at intervals of 1 month, and booster vaccinations may be givenperiodically thereafter. Alternate protocols may be appropriate forindividual patients. In immunotherapy of hyperproliferative disorders, asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-tumor immune response.Such response can be monitored by measuring the anti-tumor antibodies ina patient or by vaccine-dependent generation of cytolytic effector cellscapable of killing the patient's tumor cells in vitro. Such vaccinesshould also be capable of causing an immune response that leads to animproved clinical outcome (e.g., more frequent remissions, complete orpartial or longer disease-free survival) in vaccinated patients ascompared to non-vaccinated patients.

[1059] The vaccine according to the invention may contain a singlespecies of immunogenic minicells according to the invention or a varietyof immunogenic minicells, each of which displays a different immunogen.Additionally or alternatively, immunogenic minicells may each displayand/or express more than one iummunogen.

[1060] The summary of the invention described above is non-limiting andother features and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

EXAMPLES Example 1 Creation of a Minicell-Producing Bacterial Cell Line(MC-T7) that Expresses an Exogenous RNA Polymerase

[1061] In order to maximize the amount of RNA transcription fromepisomal elements in minicells, a minicell-producing cell line thatexpresses an RNA polymerase specific for certain episomal expressionelements was created. This E. coli strain, designated MC-T7, was createdas follows.

[1062] The P678-54 E. coli strain contains mutations that influence celldivision and induce the production of minicells (Adler et al., Proc.Natl. Acad. Sci. 57:321-326 (1967), Allen et al., Biochem. Biophys. Res.Communi. 47:1074-1079 (1972), Hollenberg et al., Gene 1:33-47 (1976)).The P678-54 strain is resistant to Lambda phage due to a mutation in themalT gene (Gottesman, Bacteriophage Lambda: The Untold Story. J. Mol.Biol. 293:177-180, 1999; Friedman, Interactions Between BacteriophageLambda and its Escherichia Coli Host. Curr. Opin. Genet. Dev. 2:727-738,1992). Thus, as an initial step, the P678-54 strain was altered so as tobe sensitive to Lambda phage so that it could form lysogens ofLambda-DE3 (see below). Wildtype MalT-encoding sequences were restoredvia a HFR (high frequency recombination) conjugation protocol using theG43 E. coli strain (CGSC stain 4928).

[1063] Recipient (P678-54) and donor (G43:BW6169) strains were grownovernight in 10 mL of LB media (10 g NaCl, 10 g select peptone 140, and5 g yeast extract in one liter dd₂O). The samples were centrifuged andthen concentrated in about 0.2 mL of LB media. The concentrated sampleswere combined and incubated with slow rotation for 30 minutes at 30° C.,and were then plated on LB agar plates that contained streptomycin (50μg/mL) and tetracycline (50 μg/mL). (Ampicillin, streptomycin,tetracycline, and all other chemicals were purchased from Sigma Chemical(St. Louis, Mo.) unless otherwise indicated.) Recipient cells wereresistant to streptomycin and donor cells were resistant totetracycline; only conjugates, which contained both resistance genes,were able to grow on the LB agar plates that contained streptomycin (50μg/mL) and tetracycline (50 μg/mL).

[1064] Putative conjugates were screened for Lambda phage sensitivityusing a cross streak technique, in which putative colonies werecross-streaked on an LB agarose plate (streptomycin, 50 μg/mL, andtetracycline, 50 μg/mL) that had been streaked with live Lambda phage.The streaked conjugate colonies were streaked perpendicular to theLambda phage streak; if a conjugate was sensitive to Lambda phageinfection then, upon contact with the Lambda phage streak, there wascell lysis and thus less or no bacterial growth. Thus, in the case ofconjugates that were sensitive to Lambda phage, there was deceasedbacterial growth “downstreak” from the phage streak.

[1065] The conjugate E. coli that were found to be sensitive to Lambdaphage infection were then used to create Lambda lysogens. Lysogenizationis a process during which Lambda phage incorporates its genome,including exogenous genes added thereto, into a specific site on thechromosome of its E. coli host cell.

[1066] The DE3 gene, which is present in the genome of the Lamda phageused to create lysogens, encodes RNA polymerase from bacteriophage T7.Lysogenation was carried out using the DE3-Lysogenation kit (Novagen,Madison, Wis.) essentially according to the manufacturer's instructions.A T7 polymerase dependent tester phage was used to confirm the presenceand expression of the DE3 gene on the bacterial chromosome. TheT7-dependent tester phage can only form plaques on a baterial known inthe presence of T7 polymerase. The phage uses a T7 promoter forexpression of its essential genes. Therefore in a plaque-forming assayonly cells which express T7 polymerase can be lysed by the tester phageand only these cells will allow for the formation of plaques. As isdescribed in more detail herein, episomal expression elements that areused in minicells may be designed such that transcription andtranslation of a cloned gene is driven by T7 RNA polymerase by utilizingexpression sequences specific for the T7 RNA polymerase.

Example 2 Cloning of Rat EDG-1 into the PCAL-C Expression Vector

[1067] Materials

[1068] Taq Polymerase, PCR Buffers, and PCR reagents were purchased fromRoche Molecular Biochemicals (Indianapolis, Ind.). All restrictionenzymes were purchased from Gibco BRL (Grand Island, N.Y.) andStratagene (La Jolla, Calif.). QIAprep mini and maxi kits, PCRpurification Kits, RNeasy miniprep kits, and the One Step RT-PCR Kitwere purchased from QIAGEN (Valencia, Calif.). The Geneclean Kit waspurchased from BIO 101 (Carlsbad, Calif.). IPTG(isopropy-beta-D-thiogalactopyranoside), T4 DNA Ligase, LB Mediacomponents and agarose were purchased from Gibco BRL. The pCAL-cprokaryote expression vector and competent cells were purchased fromStratagene.

[1069] The pCAL-c expression vector has a structure in which an ORF maybe operably linked to a high-level (but T7 RNA polymerase dependent)promoter, sequences that bind the E. coli Lac repressor, and the strongT7 gene 10 ribosome-binding site (RBS). The LacI repressor is alsoencoded by an expressed from te pCAL-c vector. As long as it is bound toits recognition sequences in the pCAL-c expression element, the lacrepressor blocks transcription from the T7 promoter. When an inducingagent, such as IPTG is added, the lac repressor is released from itsbinding sites and transcription proceeds from the T7 protmoter, providedthe T7 RNA polymerase is present. After induction, the cloned andexpressed protein may constitute the majority of newley expressedcellular proteins due to the efficient transcription and translationprocesses of the system.

[1070] Amplification

[1071] The first step in cloning rat Edg-1 (rEDG-1) into an expressionvector was to design primers for amplification via PCR (polymerase chainreaction). PCR primers were designed using the rat Edg-1 sequence(Nakajima et al., Biophy, J. 78:319A, 2000) in such a manner that theycontained either sites for NheI (GCTAGC) or BamHI (GGATCC) on their fiveprime ends. The upstream primer had the sequence of SEQ ID NO:31. Thethree prime downstream primer (SEQ ID NO:32) also contained a stopcodon, as the pCAL-c vector contains a Calmodulin Binding Protein (CBP)“tag” at its carboxyl terminus which was not intended to be incorporatedinto the rat Edg-1 polypeptide in this expression construct. The primerand resulting PCR products were designed so that the five prime end ofthe rat Edg-1 ORF was in frame with the methionine start codon found inthe pCAL-c vector.

[1072] Oligonucleotide Primer Sequences for Cloning into PCAL-CEdg1/pCAL-c construct primers: Upstream primer5′-AATTGCTAGCTCCACCAGCATCCCAGTGGTTA-3′ (SEQ ID NO: 31) Downstream primer5′-AATTGGATCC TTAAGAAGAAGAATTGACGTTT-3′ (SEQ ID NO:32) Edg1/CBP fusionconstruct primers: Upstream primer5′-AATTGCTAGCTCCACCAGCATCCCAGTGGTTA-3′ (SEQ ID NO:31) Downstream primer5′-AATTGGATCCAGAAGAAGAATTGACGTTTCCA-3′ (SEQ ID NO:33) Edg1/His6construct primers: Upstream primer5′-AATTGCTAGCTCCACCAGCATCCCAGTGGTTA-3′ (SEQ ID NO:31) Downstream primer5′-AATTGGATCC TTAATGATGATGATGATGATGAGAAGAAGAATTGACGTTTCC-3′ (SEQ IDNO:34) Edg3/rtPCR primers: Upstream primer5′-TTATGGCAACCACGCACGCGCAGG-3′ (SEQ ID NO:35) Downstream primer5′-AGACCGTCACTTGCAGAGGAC-3′ (SEQ ID NO:36) Edg3/pCAL-c constructprimers: Upstream primer 5′-AATTGCTAGCACGCACGCGCAGGGGCACCCGC-3′ (SEQ IDNO:37) Downstream primer 5′-AATTGGTACC TCACTTGCAGAGGACCCCATTCTG-3′ (SEQID NO:38) Edg3/His6 construct primers: Upstream primer5′-AATTGCTAGCACGCACGCGCAGGGGCACCCGC-3′ (SEQ ID NO:39) Downstream primer5′-AATTGGTACC TCAATGATGATGATGATGATGCTTGCAGAGGACCCCATTCTG-3′ (SEQ IDNO:16) GFP/pCAL-c construct primers: Upstream primer5′-GGTCGCCACCATGGTGAGCAA-3′ (SEQ ID NO:40) Downstream primer5′-TTAAGGATCC TTACTTGTACAGCTCGTCCAT-3′ (SEQ ID NO:41) GFP/CBP constructprimers: Upstream primer 5′-GGTCGCCACCATGGTGAGCAA-3′ (SEQ ID NO:42)Downstream primer 5′-TTAAGGATCCCTTGTACAGCTCGTCCATGCC-3′ (SEQ ID NO:43)

[1073] Notes

[1074] Restriction endonuclease sites are underlined

[1075] Stop codons are double underlined

[1076] The primers were used to amplify the rEdg-1 DNA ORF using thepolymerase chain reaction (PCR). The template used for amplification wasmRNA isolated from rat muscle tissue using the RNeasy Miniprep Kit(Qiagen) and was carried out essentially according to the manufacturer'sprotocol. Both the rtPCR and PCR amplification steps were carried out ina single reaction using the One Step RT-PCR Kit (Qiagen). The resultingrat Edg-1 PCR fragment was purified using the PCR Purification Kit(Qiagen). The amplified double stranded rEdg-1 DNA sequence containedthe NheI site at the 5-prime end and the BamHI site at the 3-prime end.This amplified rEdg-1 fragment was used for cloning into the pCAL-cexpression vector.

[1077] The pCAL-c expression vector contains NcoI, NheI, and BamHIrestriction sites in its multiple cloning site. In order to insertrEdg-1-encoding sequence into the expression vector, the rEdg-1 PCRfragment and the pCAL-c expression vector were digested with NheI andBamHI restriction enzymes for one hour at 37° C. The reaction mixturefor the digestion step consisted of 1 μg of DNA, 1×restriction buffer,and 1 μL of each enzyme. The reaction mixture was brought to a finalvolume of 20 μL with ddH2O (dd, double distilled). After 45 minutes, 1μL of Calf Intestine Alkaline Phosphatase (CIAP) was added to the pCAL-creaction mixture in order to remove the terminal phosphates from thedigested plasmid DNA. The reactions were incubated for an additional 15minutes at 37° C. The digested DNA samples were the run on a 1% TAE(Tris-acetate/EDTA electrophoresis buffer) agarose gel at 130 volts for45 minutes. The bands were visualized with UV light after the gel wasstained with ethidium bromide.

[1078] The appropriate bands were cut out of the gel for purificationusing the Geneclean Kit (BIO101). The Purified DNA fragments were thenquantified on a 1% TAE agarose gel. For the ligation reaction, ratios ofinsert to vector of 6:1 and 3:1 were used. A negative control comprisingvector only was also included in the ligation reactions. The reactionmixtures contained insert and vector DNA, 4 μL Ligase buffer, and 2 μLLigase. The reaction was brought up to a final volume of 20 μL withddH₂O. The ligation was carried out at room temperature for about 2hours. Ten (10) μL of the ligation reaction mixture was used forsubsequent transformation steps.

[1079] Ligated DNA was introduced into Epicurian Coli XL1-Blue competentcells using the heat shock transformation technique as follows. Theligation mixture was added to 100 μL of competent cells, placed on ice,and was incubated for about 30 minutes. The cells were then heat shockedat 37° C. for 1 minute and put back on ice for 2 minutes. Following heatshock, 950 μL of room temperature LB media was added to the cells andthe cells were shaken at 37° C. for 1 hour. Following the 1-houragitation the cells were pelleted for one minute at 12000 rpm in aEppendorf 5417C microcentrifuge. The supernatant was carefully pouredoff so that about 200 μL remained. The cells were then resuspended inthe remaining LB media and spread on 100×15 mm LB agarose platescontaining 50 μg/mL ampicilin. The plates were incubated overnight at37° C. Colonies were counted the following day, and the ratio ofcolonies between the negative control and the ligated samples wasdetermined. A high ratio of the number of colonies when the ligationmixture was used to transform cells, as contrasted to the number ofnegative control colonies indicated that the cloning was successful.Transformed colonies were identified, isolated, and grown overnight inLB media in the presence of ampicillin. The resulting bacterialpopulations were screened for the presence of the Edg-1-pCAL-cexpression construct.

[1080] Plasmid DNA was isolated from the cells using the QIAprep SpinMiniprep Kit (Qiagen). Isolated Edg-1-pCAL-c constructs were screenedusing the restriction enzyme ApaI, which digests the Edg-1-pCAL-cconstruct at two different sites: one in the Edg-1 coding sequence andone in the pCAL-c vector itself. The plasmid preparations were digestedwith ApaI electrophoresed on a 1% TAE agarose gel and visualized usinguv light and ethidium bromide staining. The predicted sizes of theexpected DNA fragments were 2065 bp and 4913 bp. As shown in FIG. 3,bands of the predicted size were present on the gel. The entireEdg-1-pCAL-c construct was sequenced in order to confirm its structure.This expression construct, a pCAL-c derivative that contains the ratEdg-1 ORF operably linked to a T7 promoter and lac repressor bindingsites, is designated “prEDG-1“herein.

Example 3 Construction of Rat EDG-1-CBP Fusion Protein

[1081] In order to detect rat Edg-1 protein expression, rEdg-1 codingsequences were cloned into the pCAL-c vector in frame with a CBP fusiontag. The cloning strategy for the rEdg-1-CBP construct was performedessentially as described for the Edg-1-pCAL-c construct with thefollowing differences. The PCR primers (SEQ ID NOS:3 and 5) were asdescribed for the Edg-1-pCAL-c cloning except for the omission of thestop codon in the downstream primer (SEQ ID NO:33). The removal of thestop codon is required for the construction of the Edg-1-CBP fusionprotein. The pCAL-c vector is designed so that, when the BamHI site isused for insertional cloning, and no stop codon is present in an ORFinserted into the pCAL-c expression vector the cloned ORF will bein-frame with the CBP fusion tag. Because the three prime downstreamprimer did not contain a stop codon, a CBP fusion tag could be clonedin-frame with the Edg-1 ORF. Other cloning steps were performedessentially as described before. The resulting plasmid, a pCAL-cderivative that comprises an ORF encoding a rat Edg-1-CBP fusion proteinoperably linked to a T7 promoter and lac repressor binding sites, isdesignated “prEDG-1-CBP” herein.

Example 4 Cloning of a HIS-TAGGED Rat EDG-1 into PCAL-C ExpressionVector

[1082] The rEdg-1 protein was manipulated to generate a fusion proteinhaving a 6×His tag at its carboxyl terminus. A “6×His tag” or “His tag”is an amino acid sequence consisting of six contiguous histidineresidues that can be used as an epitope for the binding of anti-6×Hisantibodies, or as ligand for binding nickel atoms. The His-tagged rEdg-1fusion protein is used to detect rEdg-1 protein expression in theminicell expression system environment.

[1083] The rEdg-1-6×His construct was cloned using the strategydescribed above for the construction of the rEdg-1-pCAL-c expressionconstruct (prEDG-1), with the upstream primer having the sequence of SEQID NO:3, but with the exception that the three prime downstream primer(SEQ ID NO:34) was designed to contain six histidine codons followed bya stop codon. The 18 base pair 6×His tag was incorporated into thecarboxyl terminus of the Edg-1 protein as expressed from the pCAL-cvector. Subsequent cloning procedures (PCR, restriction digest, gelpurification, ligation, transformation, etc.) were performed asdescribed previously for the Edg-1-pCAL-c construct (prEDG-1). Theresulting plasmid, a pCAL-c derivative that comprises an ORF encoding acarboxy-terminal His-tagged rat Edg-1-CBP fusion protein operably linkedto a T7 promoter and lac repressor binding sites, is designated“prEDG-1-6×His” herein.

Example 5 Amplification and Cloning of Rat EDG-3 Sequences

[1084] The Edg-3 full length coding sequence was amplified via PCR fromrat skeletal muscle mRNA using primers (SEQ ID NOS:35 and 36) designedfrom the known mouse sequence (Genbank accession NM_(—)010101). The mRNAused as a template for the amplification reaction was isolated using theRNeasy Miniprep Kit (Qiagen). Both the rtPCR and PCR amplification stepswere carried out in a single reaction using the One Step RT-PCR Kit(Qiagen). The rEdg-3 PCR products were visualized with UV afterelectrophoresis in 1% TAE agarose gels and ethidium bromide staining.

[1085] The predicted size of the amplified PCR products is 1145 basepairs. An appropriately-sized DNA band was isolated from the TAE gel andpurified using the Geneclean Kit (BIO101). The purified band was ligatedto the pCR3.1 vector using the TA-cloning kit (Invitrogen). Othercloning steps were carried out as described previously for the cloningof the rEdg-1-pCAL-c construct (prEDG-1) with the exception that thesamples were screened using the EcoRI restriction enzyme. The expectedsizes of the digested bands were 1145 base pairs and 5060 base pairs.Positive clones were analyzed by automated sequencing. The nucleotidesequences were analyzed using BLAST searches from the NCBI web site(www.ncbi.nlm.nih.gov/). The predicted full length rat Edg-3 amino acidsequence was assembled from the nucleotide sequencing data using insilico translation. The pCR3.1 vector comprising the rat Edg-3 ORF isdesignated “pCR-rEDG-3” herein.

Example 6 Cloning of Rat EDG-3 Coding Sequences into the PCAL-CExpression Vector

[1086] In order to express it in the minicell expression system, the ratEdg-3 ORF was cloned into the pCAL-c expression vector. The cloningstrategy used was as described above for the cloning of the rat Edg-1gene into the pCAL-c vector with the following exceptions. The primersused for PCR amplification were designed from the rat Edg-3 sequence andcontained sites for the restriction enzymes NheI and KpnI (GGTACC). TheNheI site was added to the five prime upstream primer (SEQ ID NO:37) andthe KpnI site was added to the three prime downstream primer; SEQ IDNO:38). The NheI and KpnI restriction enzymes were used for thedigestion reaction. The reaction mixture for the digestion stepconsisted of 1 μg of DNA, 1× restriction buffer (provided with theenzyme), and 1 μL of each enzyme. Plasmid preparations were screened bydigestion with NheI and KpnI. The digested plasmid DNA waselectrophosesed on a TAE agarose gel and visualized by UV after stainingwith ethidium bromide. The resultant band sizes were predicted to be1145 base pairs and 5782 base pairs. The positive plasmid clones wereanalyzed with automated sequencing. The resulting plasmid, a pCAL-cderivative that comprises an ORF encoding a rat Edg-3 protein operablylinked to a T7 promoter and lac repressor binding sites, is designated“pEDG-3“herein.

Example 7 Cloning of a His-Tagged Rat EDG-3 into the PCAL-C ExpressionVector

[1087] In order to detect expression of the rat Edg-3 protein in theminicell expression system, the rat Edg-3 coding sequence wasmanipulated so as to contain a 6×His tag at the carboxyl terminus of theprotein. The cloning strategy used to create this construct wasessentially the same as described above for the rEdg-3-pCAL-c (prEDG-3)construct cloning, with the upstream primer having the sequence of SEQID NO:37, with the exception that the three-prime downstream primer (SEQID NO:18) was designed to contain a 6×His coding sequence followed by astop codon, which allowed for the incorporation of the 6×His amino acidsequence onto the carboxyl terminus of the Edg-3 receptor protein. Othercloning and screening steps were performed as described above. Theresulting plasmid, a pCAL-c derivative that comprises an ORF encoding acarboxy-terminal His-tagged rat Edg-3 fusion protein operably linked toa T7 promoter and lac repressor binding sites, is designated“prEDG-3-6×His” herein.

Example 8 GFP Cloning into PCAL-C Expression Construct

[1088] Cloning of GFP-encoding nucleotide sequences into the pCAL-cvector was performed in order to produce an expression construct havinga reporter gene that can be used to detect protein expression (GFP,green flourescent protein). The cloning strategy used was essentiallythe same as the cloning strategy described above with the followingexceptions. The template used for PCR amplification was the peGFPplasmid “construct” (GFP construct sold by Clontech). The primers usedfor amplification were designed from the GFP coding sequence andcontained sites for the restriction enzymes NcoI and BamHI. The NcoIsite was added to the five prime upstream primer (SEQ ID NO:40) and theBamHI site was added to the three prime downstream primer; see SEQ IDNO:41) The NcoI and BamHI restriction enzymes were used for thedigestion reaction. The reaction mixture for the digestion stepconsisted of 1 μg of DNA, 1× restriction buffer (provided with theenzyme), and 1 μL of each enzyme. The screening of the plasmidpreparations was carried out using NcoI and BamHI. Digested plasmidpreparations were electrophoresed and visualized on TAE agarose gelswith UV after staining with ethidium bromide. Restriction productshaving the predicted sizes of 797 and 5782 base pairs were seen.Positive plasmid clones were sequenced using an automated sequencer. Theresulting plasmid, a pCAL-c derivative that comprises an ORF encoding arEdg-3-GFP fusion protein operably linked to a T7 promoter and lacrepressor binding sites, is designated “prEDG-3-GFP” herein.

Example 9 Design Construction of Control Expression Elements

[1089] Control expression elements used to detect and quantifyexpression of proteins in minicells were preposed. These controls directthe expression of detectable proteins. An expression element used aspositive control is pPTC12, which is supplied with the pCAL-c expressionvector from Stratagene. This construct contains an ORF encoding a fusionprotein comprising beta-galactosidase linked to CBP. Induction ofexpression of pTC12 should result in the production of a protein ofabout 120 kD, and this protein is detected via its enzymatic activity orby using antibodies directed to epitopes on the beta-galactosidase orCBP polypeptide.

[1090] A GFP fusion construct was created and used as a positive controlfor the CBP detection kit. This construct was a positive control forinduction of protein expression in the minicell expression system. Thecloning strategy used to create the construct was essentially the sameas that used for the cloning of the GFP into the pCAL-c expressionvector, with the exception that the three prime downstream primer didnot contain a stop codon; this allowed for the in frame incorporation ofthe CBP fusion tag to the GFP protein. The upstream primer had thesequence of SEQ ID NO:42, and the downstream primer had the sequence SEQID NO:43. The nucleotide sequence of the expression element wasconfirmed using an automated sequencer. The resulting plasmid, a pCAL-cderivative that comprises an ORF encoding GFP operably linked to a T7promoter and lac repressor binding sites, is designated “pGFP-CBP”herein.

Example 10 Introduction of PCAL-C Expression Constructs into the MC-T7Eschericihia Coli Strain

[1091] The MC-T7 E. coli strain was made competent using the CaCl₂technique. In brief, cells were grown in 40 mL LB medium to an OD₆₀₀ of0.6 to 0.8, and then centrifuged at 8000 rpm (7,700 g) for 5 min at 4°C. The pellet was resuspended in 20 mL of cold CaCl₂ and left on ice forfive minutes. The cells were then centrifuged at 8000 rpm (7,700 g) for5 min at 4° C. The cell pellet was resuspended in 1 mL of cold CaCl₂ andincubated on ice for 30 min. Following this incubation 1 mL of 25%glycerol was added to the cells and they were distributed and frozen in200 μL aliquots. Liquid nitrogen was used to freeze the cells. Thesecells subsequently then used for the transformation of expressionconstructs.

Example 11 Preperation of Minicells

[1092] To some degree, the preparation of minicells varied according tothe type of expression approach that is used. In general, there are twosuch approaches, although it should be noted from the outset that theseapproaches are neither limiting nor mutually exclusive. One approach isdesigned to isolate minicells that already contain an expressedtherapeutic protein or nucleic acid. Another approach is designed toisolate minicells that will express the protein or nucleic acid in theminicell following isolation.

[1093]E. coli are inoculated into bacterial growth media (e.g., Luriabroth) and grown overnight. After this, the overall protocol varies withregards to methods of induction of expression. The minicell producingcultures used to express protein post isolation are diluted and grown tothe desired OD₆₀₀ or OD450, typically in the log growth phase ofbacterial cultures. The cultures are then induced with IPTG and thenisolated. The IPTG concentration and exposure depended on whichconstruct was being used, but was usually about 500 μM final for a shorttime, typically about 4 hours. This treatment results in the productionof the T7 polymerase, which is under control of the LacUVR5 promoter,which is repressed by the LacI repressor protein. IPTG relieves the LacIrepression and thus induces expression from the LacUVR5 promoter whichcontrols expression of the T7 polymerase from the chromosome. Thispromoter is “leaky” that is, there is always a basal level of T7polymerase which can be selected for or against so that the inductionbefore isolation is not required. (This induction step is not requiredif a non-T7 expression system is used, as the reason for this step is toexpress the T7 RNA polymerase in the minicell-producing cells so thatthe polymerase and molecules segregate with the minicell.)

[1094] The E. coli cultures that produce minicells containing atherapeutic protein or nucleic acid have different induction protocols.The overnight cultures are diluted as described above; however, in thecase of proteins that are not toxic to the parent cells, this time themedia used for dilution already contains IPTG. The cultures are thengrown to mid-log growth and minicells are isolated. These culturesproduce the therapeutic protein or nucleic acid as they grow, and theminicells derived therefrom contain the therapeutic protein or nucleicacid.

[1095] Altenatively or additionally, IPTG is added and expression isinduced after the isolation of minicells. In the case of non-toxicproteins or nucleic acids that are expressed from expression elements inminicells, this treatment enhances production of the eposimally encodedgene product. In the case of toxic gene products inductionpost-isolation is preferred.

Example 12 Minicell Isolaton

[1096] Minicells were isolated from the minicell producing MC-T7 strainof E. coli using centrifugation techniques. The protocol that was usedis essentially that of Jannatipour et al. (Translocation of VibrioHarveyi N,N′-Diacetylchitobiase to the Outer Membrane of EscherichiaColi, J. Bacteriol. 169: 3785-3791, 1987) and Matsumura et al.(Synthesis of Mot and Che Products of Escherichia coli Programmed byHybrid ColE1 Plasmids in Minicells, J. Bacteriol. 132:996-1002, 1977).

[1097] In brief, MC-T7cells were grown overnight at 37° C. in 2 to 3 mLof LB media containing ampicillin (50 μg/mL), streptomycin (50 μg/mL),and tetracycline (50 μg/mL) (ampicillin was used only when growing MC-T7cells containing a pCAL-c expression construct). The cells were diluted1:100 in a total volume of 100 to 200 mL LB media with antibiotics, andgrown at 37° C. until they reached an OD₆₀₀ of 0.4 to 0.6, which isroughly beginning of the log growth phase for the MC-T7 E. coli. Duringthis incubation the remainder of the overnight culture was screened forthe presence of the correct expression construct using the techniquesdescribed above. When the cultures reached the appropriate OD₆₀₀ theywere transferred to 250 mL GS3 centrifuge bottles and centrifuged(Beckman centrifuge) at 4500 rpm (3,500 g) for 5 min. At this point thesupernatant contains mostly minicells, although a few relatively smallwhole cells may be present.

[1098] The supernatant was transferred to a clean 250 mL GS3 centrifugebottle and centrifuged at 8000 rpm (11,300 g) for 10 min. The pellet wasresuspended in 2 mL of 1×BSG (10×BSG: 85 g NaCl, 3 g KH₂PO₄, 6 gNa₂HPO₄, and 1 g gelatin in 1 L ddH₂O) and layered onto a 32 mL 20%continuous sucrose gradient. The sucrose gradient was made with sucrosedissolved in 1×BSG.

[1099] The sucrose gradient was then loaded in a Beckman SW24 rotor andcentrifuged in a Beckman Ultracentrifuge at 4500 rpm (9,000 g) for 14min. Following ultracentrifugation a single diffuse band of minicellswas present. The top two thirds of this band was aspirated using a 10 mLpipette and transferred to a 30 mL Oakridge tube containing 10 mL of1×BSG. The sample was then centrifuged at 13,000 rpm (20,400 g) for 8min. Following centrifugation, the pellet was resuspended in 2 mL 1×BSG,and the resuspended cells were loaded onto another 5 to 20% sucrosegradient. This sucrose gradient was centrifuged and the minicells werecollected as described above. The sucrose gradient procedure wasrepeated a total of three times.

[1100] Following the final sucrose gradient step the entire minicellband was collected from the sucrose gradient and added to a 30 mLOakridge tube that contained 10 mL of MMM buffer (200 mL 1×M9 salts, 2mL 20% glucose, and 2.4 mL DIFCO Methionine Assay Medium). This minicellsolution was centrifuged at 13,000 rpm (20,400 g) for 8 min. The pelletwas resuspended in 1 mL of MMM Buffer.

[1101] The concentration of minicells was determined using aspectrophotometer. The OD₄₅₀ was obtained by reading a sample ofminicells that was diluted 1:100.

Example 13 Other Methods to Prepare and Isolate Minicells

[1102] By way of non-limiting example, induction of E. coli parentalcells to form minicells may occur by overexpression of the E. coli ftsZgene. To accomplish this both plasmid-based and chromosomaloverexpression constructs were created that place the ftsZ gene underthe control of various regulatory elements (Table 6). TABLE 6 REGULATORYCONSTRUCTS CONTROLLING FTSZ EXPRESSION. Regulatory region inducer[inducer] SEQ ID NO.: Para::ftsZ Arabinose 10 mM 1, 3 Prha::ftsZRhamnose 1 mM 2, 4 Ptac::ftsZ IPTG 30 μM       5, Garrido et al.^(a)  

[1103] Oligonucleotide Names and PCR Reactions Use the Following Format

[1104] “gene-1” is N-terminal, 100% homology oligo for chromosomal orcDNA amplification

[1105] “gene-2” is C-terminal, 100% homology oligo for chromosomal orcDNA amplification

[1106] “gene-1-RE site” is same sequence as gene-1 with additionalresidues for remainder of sequence, RE sites, and/or chimeric fusions.

[1107] “gene-2-RE site” is same sequence as gene-1 with additionalresidues for remainder of sequence, RE sites, and/or chimeric fusions.

[1108] Use “gene-1, 2” combo for chromosomal/cDNA amplification and“gene-1 RE site, gene-2-RE site” to amplify the mature sequence from the“gene-1, 2” gel-purified product. TABLE 7 OLIGONUCLEOTIDE PRIMERSEQUENCES FOR TABLE 6 CONSTRUCTS SEQ ID NO.: Primer name 5′to 3′sequence44 FtsZ-1 CCAATGGAACTTACCAATGACGCGG 45 FtsZ-2 GCTTGCTTACGCAGGAATGCTGGG46 FtsZ-1-PstI CGCGGCTGCAGATGTTTGAACCAATGGAACTTACCAATGAC GCGG 47FtsZ-2-XbaI GCGCCTCTAGATTATTAATCAGCTTGCTTACGCAGGAATGC TGGG

[1109] Table 7 oligonucleotide sequences are for use in cloning ftsZinto SEQ ID NO.:1 and 2 (insertions of ftsZ behind the arabinosepromotor (SEQ ID NO.: 1) and the rhamnose promotor (SEQ ID NO.: 2).TABLE 8 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR FTSZ CHROMOSOMALDUPLICATION CONSTRUCTS SEQ ID NO.: Primer name 5′to 3′sequence 48 Kan-1GCTAGACTGGGCGGTTTTATGGACAGCAAGC 49 Kan-2GCGTTAATAATTCAGAAGAACTCGTCAAGAAGGCG 50 Kan-1-X-frtGCGCCTACTGACGTAGTTCGACCGTCGGACTAGCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGCTAGACTGG GCGGTTTTATGGACAGCAAGC 51Kan-2-intD-frt CAAGATGCTTTGCCTTTGTCTGAGTTGATACTGGCTTTGGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGCGTT AATAATTCAGAAGAACTCGTCAAGAAGGCG52 AraC-1 CGTTACCATTATGACAACTTGACGG 53 RhaR-1TTAATCTTTCTGCGAATTGAGATGACGCC 54 LacI^(q)-1 GTGAGTCGATATTGTCTTTGTTGACCAG55 Ara-1-intD GCCTGCATTGCGGCGCTTCAGTCTCCGCTGCATACTGTCCCGTTACCAATTATGACAACTTGACGG 56 RhaR-1-intDGCCTGCATTGCGGCGCTTCAGTCTCCGCTGCATACTGTCC TTAATCTTTCTGCGAATTGAGATGACGCC57 LacI^(q)-1-intD GCCTGCATTGCGGCGCTTCAGTCTCCGCTGCATACTGTCCTTAATAAAGTGAGTCGATATTGTCTTTGTTGACCAG 58 FtsZ-1-XGCCTGCATTGCGGCGCTTCAGTCTCCGCTGCATACTGTCC CGTTACCAATTATGACAACTTGACGG

[1110] In like fashion, the ftsZ gene was amplified from SEQ ID NO.: 1,2 and Ptac::ftsZ (Garrido, T. et al. 1993. Transcription of ftsZoscillates during the cell cycle of Escherichia coli. EMBO J.12:3957-3965) plasmid and chromosomal constructs, respectively using thefollowing oligonucleotides:

[1111] For amplification of araC through ftsZ of SEQ ID NO.: 1 useoligonucleotides:

[1112] AraC-1

[1113] FtsZ-2

[1114] For amplification of rhaR through ftsZ of SEQ ID NO.: 2 useoligonucleotides:

[1115] RhaR-1

[1116] FtsZ-2

[1117] For amplification of laclI^(q) through ftsZ of Ptac::ftsZ(Garrido, T., et al.) use oligonucleotides:

[1118] lacI^(q)-1

[1119] ftsZ-2

[1120] The above amplified DNA regions were gel-purified and used astemplate for the second round of PCR using oligonucleotides containinghomology with the E. coli chromosomal gene intd and on the other endwith random sequence termed “X”. Oligonucleotides used in this round ofPCR are shown below:

[1121] For amplification of araC through ftsZ from SEQ ID NO.: 1 tocontain homology to intD and the random X use oligonucleotides:

[1122] AraC-1-intD

[1123] FtsZ-1-X

[1124] For amplification of rhaR through ftsZ from SEQ ID NO.: 2 tocontain homology to intD and the random X use oligonucleotides:

[1125] RhaR-1-intD

[1126] FtsZ-1-X

[1127] For amplification of lacIq through ftsZ from Ptac::ftsZ tocontain homology to intd and the random X use oligonucleotides:

[1128] LacIq-1-intD

[1129] FtsZ-1-X

[1130] The PCR products from these PCR reactions are as shown below:

[1131] intD-araC-Ara promotor-ftsZ-“X”

[1132] intd-rhaRS-Rha promotor-ftsZ-“X”

[1133] intD-lacI^(q)-Ptac promotor-ftsZ-“X”

[1134] To amplify the mature complexes, the following regions were mixedand amplified with the coupled oligonucleotide sequence primers:

[1135] These expression constructs may be expressed from the plasmid,placed in single copy, replacing the native ftsZ copy on the E. colichromosome (Garrido, T., et al. 1993. Transcription of ftsZ oscillatesduring the cell cycle of Escherichia coli. EMBO J. 12:3957-3965), or induplicate copy retaining the native ftsZ copy while inserting one of theexpression constructs in Table 6 into the intD gene on the samechromosome. Chromosomal duplications were constructed using the REDrecombinase system (Katsenko, K. A., and B. L. Wanner. One-StepInactivation of Chromosomal Genes in Escherichia coli K-12 Using PCRProducts. Proc. Natl. Acad. Sci. 97:6640-6645. 2000) and are shown inSEQ ID NO 3-5. The later constructs allow native replication duringnon-minicell producing conditions, thus avoiding selective pressureduring strain construction and maintenance. Furthermore, these strainsprovide defined points of minicell induction that improve minicellpurification while creating conditions that allow strain manipulationprior to, during, and following minicell production. By way ofnon-limiting example these manipulations may be protein production thatthe cytoplasmic redox state, modify plasmid copy number, and/or producechaperone proteins.

[1136] For minicell production, a minicell producing strain described inthe previous section is grown overnight in Luria broth (LB) supplementedwith 0.1% dextrose, 100 μg/ml ampicillin, and when using the single-copyftsZ construct, 15 μM IPTG. All incubations were performed at 37° C. Forminicell induction only, overnight strains are subcultured 1/1000 intothe same media. If minicell induction is to be coupled withco-expression of other proteins that are controlled by a cataboliterepression-sensitive regulator, dextrose was excluded. Minicellinduction is sensitive to aeration and mechanical forces. Therefore,flask size, media volume and shake speed is critical for optimal yields.Likewise, bioreactor conditions must be properly regulated to optimizethese production conditions.

[1137] In shake-flask cultures, strains are grown to early exponential(log) phase as monitored by optical density (OD) at 600 nm (OD₆₀₀0.05-0.20). (Bioreactor conditions may differ significantly depending onthe application and yield desired). For minicell induction alone, earlylog phase cultures are induced with the appropriate inducerconcentration shown in Table 6. For coupled co-expression, thesecultures are induced as shown in Table 6 for the appropriate minicellregulator, while the coupled protein(s) is induced with the inducerappropriate for the regulator controlling the synthesis of that protein.Cultures are grown under the appropriate conditions and harvested duringlate log (OD₆₀₀ 0.8-1.2). Depending on the application, minicell inducedcultures may be immediately chilled on ice prior to purification, ormaintained at room temperature during the harvesting process.

[1138] To separate minicells from viable, parental cells, cultures aresubjected to differential centrifugation (Voros, J., and R. N. Goodman.1965. Filamentous forms of Erwinia amylovora. Phytopathol. 55:876-879).Briefly, cultures are centrifuged at 4,500 rpm in a GSA rotor for 5 min.Supernatants are removed to a fresh bottle and centrifuged at 8,000 rpmfor an additional 10 min to pellet minicells. Pelleted minicells(containing contaminating parental cells) are resuspended in 2 ml LB,LBD (LB supplemented with 0.1% dextrose), Min (minimal M63 salt media)(Roozen, K. J., et al. 1971. Synthesis of ribonucleic acid and proteinin plasmid-containing minicells of Escherichia coli K-12. J. Bacteriol.107:21-23), supplemented with 0.5% casamino acids) or MDT (minimal M63salt media, supplemented with 0.5% casamino acids, 0.1% dextrose, andthiamine). Resuspended minicells are next separated using linear densitygradients. By way of non-limiting example, these gradients may containsucrose (Cohen A., et al. 1968. The properties of DNA transferred tominicells during conjugation. Cold Spring Harb. Symp. Quant. Biol.33:635-641), ficol, or glycerol. For example, linear sucrose gradientsrange from 5-20% and are poured in LB, LBD, Minor MDT. Using a SW28swinging bucket rotor, gradients are centrifuged at 4,500 rpm for 14min. Banded minicells are removed, mixed with LB, LBD, Minor MDT, andusing a JA-20 rotor are centrifuged at 13,000 rpm for 12 min. Followingcentrifugation, pellets are resuspended in 2 ml LB, LBD, Minor MDT andsubjected to a second density gradient. Following the second densityseparation, banded minicells are removed from the gradient, pelleted asdescribed, and resuspended in LB, LBD, Minor MDT for use and/or storage.

[1139] Purified minicells are quantitated using an OD₆₀₀ measurement ascompared to a standard curve incorporating LPS quantity, minicell size,and minicell volume. Quantitated minicells mixtures are analyzed forcontaminating, viable parental cells by plating on the appropriategrowth media (Table 9). TABLE 9 MINICELL PURIFICATION AND PARENTAL CELLQUANTITATION Purifi- Total Fold- cation Total cells parental cells MC/PCratio purification Before 4.76 × 10¹¹ 3.14 × 10¹¹ 0.25/1 — After 1.49 ×10¹¹ 6.01 × 10⁴  2.48 × 10⁶/1 5.23 × 10⁶

Example 14 Protoplast Formation

[1140] In order to allow a membrane receptor to be presented to theoutside environment (displayed), minicells are made into protoplasts. Inorder to make the integral membrane protein receptors in the innermembrane more accessible for ligand binding, the outer membrane and cellwall were removed. The removal of the outer membrane and cell wall fromE. coli whole cells and minicells to produce protoplasts was performedessentially according to previously described protocols with a fewmodifications (Birdsell et al., Production and Ultrastructure ofLysozyme and Ethylenediaminetetraacetate-Lysozyme Spheroplasts ofEscherichia coli, J. Bacteriol. 93:427-437, 1967; Weiss et al.,Protoplast Formation in Escherichia Coli, J. Bacteriol. 128:668-670,1976. Both minicells and whole cells were processed the same way.

[1141] In brief, the cells were grown to mid-log phase and pelleted atroom temperature (minicells were isolated from cultures in mid-logphase). The pellet was washed twice with 10 mM Tris. Following thesecond wash protoplast production may be performed using two approaches.In the first approach, following the second wash, the cells wereresuspended in 100 mM Tris (pH 8.0) that contained 6-20% sucrose and putin a 37° C. waterbath (the Tris/sucrose buffer was pre-warmed to 37°C.). The volume used to resuspend the cells was determined by thefollowing equation: (volume of cells×OD₄₅₀)/10=resuspension volume.After a 1 minute incubation, 2 mg/mL lysozyme was added to a finalconcentration of 5-100 μg/mL. The samples were then incubated for 12minutes at 37° C. while being gently mixed. Next, 100 mM EDTA (pH 7) wasslowly added over a period of 2.5 minutes (amount of EDTAadded=1/00-1/10 volume of cells) followed by a 10 min incubation at 37°C. The protoplasts are also diluted from 20% sucrose down to either 10%or 5% sucrose, which facilitates the complete removal of the outermembrane and cell wall. The protoplasts thus generated were separatedfrom the outer membrane and cell wall using a sucrose step gradient. Asucrose step gradient does not have a gradual increase in sucrosepercentage; rather, it goes directly from one percent to the other. Forexample, protoplasts generated from whole cells are loaded on a stepgradient that is made from 5% and 15% sucrose. The protoplasts spinthrough the 15% sucrose but the debris generated when making theprotoplasts does not spin through the 15% sucrose. The protoplasts arethus separated from the debris. The second method to prepareprotoplasts, following the second wash, I×10⁹ cells were resuspendedwith 50 mM Tris, pH 8.0 containing 0.5-50 mM EDTA and 6-20% sucrose.This mixture was incubated at 37° C. for 10 min. Following incubation,the mixture was centrifuged at 13,200 RPM in a microcentrifuge for 2min. After centrifugation, the pellet was resuspened in 50 mM Tris, pH8.0 containing 5-100 μg/ml lysozyme and 6-20% sucrose. This mixture wasincubated at 37° C. for 10 min. Following incubation, the mixture wascentrifuged at 13,200 RPM in a microcentrifuge for 2 min, resuspended in50 mM Tris pH 8.0 containing 6-20% sucrose for use.

[1142] An alternative method to remove contaminating LPS is to useaffinity absorption with an anti-LPS antibody (Cortex). To accomplishthis, the anti-LPS antibody was coated on either an activated agarose orsepharose matrix (Sigma) or epoxy-coated magnetic M-450 beads (Dynal).The spheroplast/protoplast mixture was subjected to the antibody coatedmatrix either in batch or using column chromatographic techniques toremove contaminating LPS. Following exposure, the unbound fraction(s)was collected and re-exposed to fresh matrix. To monitor the efficiencyof the protoplasting reaction and LPS removal, three constructs wereused (Table 10). TABLE 10 PROTOPLAST MONITORING CONSTRUCTS SEQ ID SEQ IDInducible Construct NO Plasmid NO protein Inducer PMPX-5 6 pMPX-32 7ΔphoA Rhamnose PMPX-5 6 pMPX-53 8 phoA Rhamnose PMPX-5 6 pMPX-33 9toxR-phoA Rhamnose

[1143] TABLE 11 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE 10 CONSTRUCTSSEQ ID NO.: Primer name 5′to 3′sequence 59 ΔphoA-1GCCTGTTCTGGAAAACCGGGCTGCTCAGGG 60 ΔphoA-2 GCGGCTTTCATGGTGTAGAAGAGATCGG61 ΔphoA-1-PstI CCGCGCTGCAGATGCCTGTTCTGGAAAACCGGGCTGCTCAG GG 62ΔphoA-2-XbaI GCGCCTCTAGATTATTATTTCAGCCCCAGAGCGGCTTTCATG GTGTAGAAGAGATCGG63 PhoA-1 GTCACGGCCGAGACTTATAGTCGC 64 PhoA-2GCGGCTTTCATGGTGTAGAAGAGATCGG 65 PhoA-1-PstICCGCGCTGCAGATGTCACGGCCGAGACTTATAGTCGC 66 PhoA-2-XbaIGCGCCTCTAGATTATTATTTCAGCCCCAGAGCGGCTTTCATG GTGTAGAAGAGATCGG 67T-phoA-1-PstI CCGCGCTGCAGATGAACTTGGGGAATCGACTGTTTATTCTGATAGCGGTCTTACTTCCCCTCGCAGTATTACTGCTCATGCCTG TTCTGGAAAACCGGGCTGCTCAGGG 68T-phoA-2-XbaI GCGCCTCTAGATTATTATTTCAGCCCCAGAGCGGCTTTCATGGTGTAGAAGAGATCGG

[1144] Oligonucleotides SEQ ID NOS.:59, 60, 61 and 62 were used toamplify phoA lacking a leader sequence (ΔphoA) form the E. colichromosome. Once amplified, this region was inserted into SEQ ID NO.: 6using PstI and XbaI to create SEQ ID NO.: 7.

[1145] Oligonucleotides SEQ ID NOS.:63, 64, 65 and 66 were used toamplify phoA containing a leader sequence (phoA) form the E. colichromosome. Once amplified, this region was inserted into SEQ ID NO.: 6using PstI and XbaI to create SEQ ID NO.: 8.

[1146] Oligonucleotides SEQ ID NOS.:59, 60, 67 and 68 were used toamplify phoA lacking a leader sequence (ΔphoA) form the E. colichromosome and form a translational fusion between the transmembranedomain of toxR from Vibrio cholerae. Once amplified, this region wasinserted into SEQ ID NO.: 6 using PstI and XbaI to create SEQ ID NO.: 9.

[1147] By co-expression of minicells and protein, minicells wereprepared that contained cytoplasmic PhoA (pMPX-32 expresses phoA lackinga leader sequence [ΔphoA]), periplasmic PhoA (pMPX-53 expresses nativephoA that exports to the periplasmic space), or inner membrane-boundPhoA (pMPX-33 expresses phoA lacking a leader sequence fused to thetransmembrane domain (TMD) of the toxR gene product from Vibriocholerae). Using these expressed proteins, the efficiency of minicellprotoplasting was monitored (Table 12). TABLE 12 EFFICIENCY OF MINICELLPROTOPLAST PREPARATION AND PURIFICATION Step Location ^(a) ΔPhoA PhoAT-PhoA LPS total ^(b) Minicell Pellet 100 100 100 100 EDTA/lysozymeWhole 100 100 100 100 1^(st) Anti-LPS Pellet 80 0 80 30 2^(nd) Anti-LPSPellet 60 0 60 0

[1148] The data suggests that periplasmic PhoA is lost during thepreparation, while both cytoplasmic and membrane-bound PhoA are retainedin a cellular body that lacks LPS. However, during this process ˜40% ofthe total minicell content is lost.

Example 15 T7-Dependent Induction of Expression

[1149] Expression from the pCAL-c expression vector is driven from a T7bacteriophage promoter that is repressed by the LacI gene product.Transcription of the DNA into mRNA, and subsequent translation of mRNAinto proteins, does not occur as long as the LacI repressor is bound tothe T7 promoter. However, in the presence of IPTG, the LacI repressordoes not bind the T7 promoter. Thus, induction of expression from pCAL-csequences is dependent on the presence of IPTG. Slightly differentprotocols were used for the induction of Escherichia coli whole and forthe induction of minicells. Slight differences are also present in theprotocols for induction of minicells for ³⁵S-methionine labeling ofproteins in contrast to those for the induction of minicells for Westernblot analysis. These induction protocols are described bellow.

[1150] For expression in E. coli whole cells, the cells were first grownovernight in 3 mL of LB and antibiotics. The cultures were screened forthe presence of the desired expression element as previously described.Cultures containing the desired expression elements were diluted 1:100and grown to an OD₆₀₀ of between 0.4 to 0.6. The culture size varieddepending on the intended use of the cells. IPTG was then added to afinal concentration of 200 μg/mL, and the cells were shaken at 30° C.for 4 hours. Following the induction, cells were harvested for analysis.

[1151] The induction of minicells was carried out as follows. Theminicells were diluted in MMM buffer to 1 mL total volume according tothe concentration obtained from the isolation procedure (OD₄₅₀ of about0.5). The cells were then treated with 50 μg/mL of cycloserine for 30minutes at 37° C. to stop whole cell growth. Following the cycloserinetreatment the cells were provided with an amino acid, methionine, whichthe MMM buffer does not contain. For ³⁵S-labeled protein induction³⁵S-methionine was added to the minicell sample whereas, for unlabeledprotein induction unlabeled methionine was added. Fifteen (15) μCi of³⁵S-methionine (Amersham Pharmacia Biotech, Piscataway, N.J.) was addedto the samples for radiolabeling and 5 μmol of methionine was added tothe non-labeled minicell samples. Two hundred (200) μg/mL IPTG was alsoadded to the minicell samples, which were then shaken at 30° C. forabout 4 hours. Following induction, the minicells were harvested forfurther preparation or analysis.

Example 16 Western Blot Analysis

[1152] The CBP detection kit was purchased from Stratagene. SDS runningbuffer, 10% Tris-HCl ready gels, Kaleidoscope Pre-stained Standards, andLaemmli Sample Buffer were purchased from BIO RAD (Hercules, Calif.).GFP (FL) HRP antibodies were purchased from Santa Cruz Biotechnology(Santa Cruz, Calif.). Edg-3CT antibody an antibody directed to thecarboxy terminus of was purchased from Exalpha Biologicals (Boston,Mass.). Anti-6×His antibody, positrope, and the WesternBreeze Kit werepurchased from Invitrogen (Carlsbad, Calif.). Protocols were carried outessentially according to the manufacturer's instructions unlessotherwise indicated.

[1153] Three different Western blot protocols were used to detectprotein expression in both a minicell expression system and in a wholecell expression system. For both systems, the SDS-PAGE gel and thetransfer protocols were essentially as follows. The samples weredenatured by diluting the samples 1:1 in Laemmli buffer (BIORAD) andthen sonicated for 10 min. The denatured samples were loaded onto a 10%Tris-Glycine gel (BIORAD) and electrophoresed at 130 V for about 1.5hours in 1×SDS running buffer (BIORAD). The electrophoresed proteinswere electrotransferred to nitrocellulose membranes at 0.5 Amps for 1.5hours in Transfer Buffer (5.8 g Tris, 2.9 g glycine, 200 mL methanol,and 3.7 mL of 10% SDS). The nitrocellulose membranes comprising thetransferred proteins were used for Western bloting.

[1154] GFP Western blots were carried out as follows. The nitrocellulosemembrane was blocked for 2 hours with 5% milk in PBST (PBS buffer with0.05% Tween). Following the blocking step the nitrocellulose membranewas washed twice with PBST. For the detection of GFP protein, ananti-GFP-HRP conjugated antibody (Santa Cruz Biotechnology) was used ata dilution of 1:3000 in PBST (HRP, horse radish peroxidase). Thenitrocellulose membrane was incubated in the anti-GFP-HRP antibodysolution for one hour and then washed twice with PBST. GFP proteins onthe nitrocellulose membrane were detected and visualized using the ECLsystem (Amersham).

[1155] The His-tagged Edg-1 and Edg-3 proteins were detected using amouse anti-6×His antibody from Invitrogen and the WesternBreezechemoluminecent Kit (Invitrogen). The antibody was diluted 1:4000 inbuffers provided by the WesternBreeze Kit. The WesternBreeze immunoblotwas carried out essentially according to the manufacturer's protocol.The Edg-1-CBP and GFP-CBP fusion proteins were detected using the CBPdetection Kit (Stratagene). All antibodies and substrates were providedin the Kit. FIG. 3 is a photo of the Western hybridization resultsshowing the presence of Edg-1-6×His and Edg-3-6×His in minicells andparent cells.

Example 17 Methods to Induce Expression

[1156] Expression in minicells may proceed following purification ofminicells and/or minicell protoplasts from parental cells and LPSconstituents, repectively. However, for some applications it is suitableto co-express proteins of interest with minicell induction. For theseapproaches, one may use the protocol described in Example 13 forexpression of the phoA constructs. By way of non-limiting example,either of these approaches may be accomplished using one or more of thefollowing expression constructs (Table 13). TABLE 13 EXPRESSIONCONSTRUCTS Regulatory SEQ ID Plasmid element(s) inducer Plasmid NO.:pMPX-5 rhaRS Rhamnose pUC-18 6 pMPX-7 uidR β-glucuronate pUC-18 10pMPX-8 melR Melibiose pUC-18 11 pMPX-18 araC Arabinose pUC-18 12 pMPX-6araC Arabinose pUC-18 13

[1157] TABLE 14 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE 13 CONSTRUCTSSEQ ID NO.: Primer name 5′to 3′sequence 69 Rha-1GCGAATTGAGATGACGCCACTGGC 70 Rha-2 CCTGCTGAATTTCATTAACGACCAG 71Rha-1-HindIII CGGCGAAGCTTAATTAATCTTTCTGCGAATTGAGATGACGC CACTGGC 72Rha-2-PstI CGCCGTAATCGCCGCTGCAGAATGTGATCCTGCTGAATTTC ATTAACGACCAG 73Uid-1 CGCAGCGCTGTTCCTTTGCTCG 74 Uid-2 CCTCATTAAGATAATAATACTGG 75Uid-1-HindIII GCCGCAAGCTTCGCAGCGCTGTTCCTTTGCTCG 76 Uid-2-PstICCAATGCATTGGTTCTGCAGGACTCCTCATTAAGATAATAA TACTGG 77 Mel-1CGTCTTTAGCCGGGAAACG 78 Mel-2 GCAGATCTCCTGGCTTGC 79 Mel-1-HindIIIGCCGCAAGCTTCGTCTTTAGCCGGGAAACG 80 Mel-2-SalI CGGTCGACGCAGATCTCCTGGCTTGC81 Ara-1 CAAGCCGTCAATTGTCTGATTCG 82 Ara-2 GGTGAATTCCTCCTGCTAGCCC 83Ara-1-HindIII GCGCCAAGCTTCAAGCCGTCAATTGTCTGATTCG 84 Ara-2-PstICTGCAGGGTGAATTCCTCCTGCTAGCCC 85 Ara-1-XhoIGCTTAACTCGAGCTTAATAACAAGCCGTCAATTGTCTGATTC 86 Ara-2-SstIGCTTAACCGCGGGCCAAGCTTGCATGCCTGCTCC

[1158] Oligonucleotides SEQ ID NOS.:69, 70, 71 and 72 were used toamplify the rhaRS genes and their divergent control region from the E.coli chromosome. Once amplified, this region was inserted into pUC18using HindIII and PstI to create SEQ ID NO.: 6.

[1159] Oligonucleotides SEQ ID NOS.:73, 74, 75 and 76 were used toamplify the uidR control region, the uidR gene and the control regionfor expression from the E. coli chromosome. Once amplified, this regionwas inserted into pUC18 using HindIII and PstI to create SEQ ID NO.: 10.

[1160] Oligonucleotides SEQ ID NOS.:77, 78, 79 and 80 were used toamplify the melR gene and its divergent control region from the E. colichromosome. Once amplified, this region was inserted into pUC18 usingHindIII and SalI to create SEQ ID NO.: 11.

[1161] Oligonucleotides SEQ ID NOS.:81, 82, 83 and 84 were used toamplify the araC gene and its divergent control region from the E. colichromosome. Once amplified, this region was inserted into pUC18 usingHindIII and PstI to create SEQ ID NO.: 12.

[1162] Oligonucleotides SEQ ID NOS.:81, 82, 85 and 86 were used toamplify the araC gene and its divergent control region was PCR amplifiedfrom pBAD-24. Once amplified, this region was inserted into pEGFP(Clontech) using XhoI and SstI to create SEQ ID NO.: 13.

[1163] Except of pMPX-6, these expression constructs contain the samemultiple cloning site. Therefore, any protein of interested may beinserted in each modular expression construct for simple expressionscreening and optimization.

[1164] By way of non-limiting example, other proteins that may beexpressed are listed in Table 15. TABLE 15 OTHER EXPRESSED PROTEINS SEQID Protein Origin Construct Purpose NO.: Edg3 Rat native GPCR 14 β2ARHuman native GPCR 15 TNFR-1a Human residues 29-455 Receptor 18 (human)TNFR-1b Human residues 41- Receptor 17 (human) 455 TNF (human) Humannative Gene transfer 19 T-EGF Human chimera Gene transfer 20 T-InvasinY. pseudotuberculosis chimera Gene transfer 21

[1165] TABLE 16 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE 15 SEQ IDNO.: Primer name 5′to 3′sequence 87 Edg-1 GGCAACCACGCACGCGCAGGGCCACC 88Edg-2 CAATGGTGATGGTGATGATGACCGG 89 Edg-1-SalICGCGGTCGACATGGCAACCACGCACGCGCAGGGCCACC 90 Edg-2-KpnIGCGCCGGTACCTTATCAATGGTGATGGTGATGATGACCG G 91 β2AR-1GGGGCAACCCGGGAACGGCAGCGCC 92 β2AR-2 GCAGTGAGTCATFTTGTACTACAATTCCTCC 93β2AR-1-SalI CGCGGTCGACATGGGGCAACCCGGGAACGGCAGCGCC 94 β2AR-2-BamHIGCGCCGGATCCTTATTATAGCAGTGAGTCATTTGTACTAC AATTCCTCC 95 TNFR(29)-1GGACTGGTCCCTCACCTAGGGGACAGGG 96 TNFR(29)-2 CTGAGAAGACTGGGCGCGGGCGGGAGG97 TNFR(29)-1-SalI CGCGGGTCGACATGGGACTGGTCCCTCACCTAGGGGACA GGG 98TNFR(29)-2-KpnI GCGCCGGTACCTTATTACTGAGAAGACTGGGCGCGGGCG GGAGG 99TNFR(41)-1 GATAGTGTGTGTCCCC 100 TNFR(41)-2 CTGAGAAGACTGGGCGC 101TNFR(41)-1-NcoI GGGAGACCATGGATAGTGTGTGTCCCC 102 TNFR(41)-2-XbaIGCCTCATCTAGATTACTGAGAAGACTGGGCGC 103 TNF-1 GAGCACTGAAAGCATGATCCGGGACG104 TNF-2 CAGGGCAATGATCCCAAAGTAGACCTGC 105 TNF-1-EcoRICCGCGGAATTCATGAGCACTGAAAGCATGATCCGGGACG 106 TNF-2-HindIIIGGCGCAAGCTTATCACAGGGCAATGATCCCAAAGTAGAC CTGC 107 T-EGF-1TCTGATAGCGGTCTTACTTCCCCTCGCAGTATTACTGCTCAATAGTGACTCTGAATGTCCCCTGTCCCACGATGGGTACT GCCTCCATGATGGTGTGTGCATGTATATTG108 T-EGF-2 AGGTCTCGGTACTGACATCGCTCCCCGATGTAGCCAACAACACAGTTGCATGCATACTTGTCCAATGCTTCAATATACA TGCACACACCATCATGGAGGCA 109T-EGF-3 CCGCGGGTACCATGAACTTGGGGAATCGACTGTTTATTCT GATAGCGGTCTTACTTCCCCTCG110 T-EGF-4 GCGCCAAGCTTATTAGCGCAGTTCCCACCACTTCAGGTCTCGGTACTGACATCGCTCCCCG 111 Inv-1 TCATTCACATTGAGCGTCACCG 112 Inv-2TTATATTGACAGCGCACAGAGCGG 113 Inv-1-ToxR-EcoRIGCAAGAATTCACCATGAACTTGGGGAATCGACTTTATTCTGATAGCGGTCTTACTTCCCCTCGCAGTATTAGTGCTCT CATTCACATTGAGCGTCACCG 114Inv-2-PstI CGCGGTTACGTAAGCAACTGCAGTTATATTGACAGCGCA CAGAGCGG

[1166] Oligonucleotides SEQ ID NOS.:87, 88, 89 and 90 were used toamplify rat Edg3 from rat cDNA. Once amplified, this region was insertedinto SEQ ID NO.: 6 (pMPX-5) using SalI and KpnI to create SEQ ID NO.:14.

[1167] Oligonucleotides SEQ ID NOS.:91, 92, 93 and 94 were used toamplify human β2 adrenergic receptor (β2AR) from human heart cDNA. Onceamplified, this region was inserted into SEQ ID NO.: 6 (pMPX-5) usingSalI and BamHI to create SEQ ID NO.:15.

[1168] Oligonucleotides SEQ ID NOS.:95, 96, 97 and 98 were used toamplify human tumor necrosis factor receptor (TNFR residues 29-455) fromhuman Jurkat CL71 cDNA. Once amplified, this region was inserted intoSEQ ID NO.: 12 (pMPX-18) using SalI and KpnI to create SEQ ID NO.:18.

[1169] Oligonucleotides SEQ ID NOS.:99, 100, 101 and 102 were used toamplify human tumor necrosis factor receptor (TNFR residues 41-455) fromhuman Jurkat CL71 cDNA. Once amplified, this region was inserted intopBAD24 using NcoI and XbaI to create SEQ ID NO.:17.

[1170] Oligonucleotides SEQ ID NOS.:103, 104, 105 and 106 were used toamplify human tumor necrosis factor (TNF) from human Jurkat CL71 cDNA.Once amplified, this region was inserted into SEQ ID NO.: 13 (pMPX-6)using EcoRI and HindIII to create SEQ ID NO.:19. TABLE 17 PROGRAM TOANNEAL GRADIENT PCR WITH PFX POLYMERASE Temp Time Step (° C.) (min) 1 952.0 2 95 0.5 3 64 0.5 4 68 2.5 5 Goto 2, 2× 6 95 0.5 7 62 0.5 8 68 2.5 9Goto 6, 4× 10 95 0.5 11 60 0.5 12 68 2.5 13 Goto 10, 6× 14 95 0.5 15 580.5 16 68 2.5 17 Goto 14, 24× 18 4 hold 19 end

[1171] Oligonucleotides SEQ ID NOS.:107, 108, 109 and 110 were mixed andPCR amplified using anneal gradient PCR (Table 17) to form mature humanepidermal growth factor (EGF) (residues 971-1023) translationally fusedto the transmembrane domain of toxR from Vibrio cholerae. Onceamplified, region was inserted into SEQ ID NO.: 13 (pMPX-6) using KpnIand HindIII to create SEQ ID NO.:20.

[1172] Using PFX polymerase (Invitrogen) oligonucleotide SEQ ID NO.:111,112, 113 and 114 were used to amplify invasin residues 490-986 (inv)from Yersinia pseudotuberculosis chromosomal DNA and form atranslational fusion between the transmembrane domain of toxR fromVibrio cholerae. Once amplified, this region was inserted into SEQ IDNO.:13 (pMPX-6) using EcoRI and PstI to create SEQ ID NO.:21.

[1173] These proteins were proof-of-principle constructs used toevaluate the minicell platform. For purposes of this initial evaluation,all proteins except TNF, T-EGF and T-Invasin were cloned into pMPX-5,with these later proteins cloned into pMPX-6 for gene transferexperiments.

[1174] Whether the approach for protein expression is co-expression withminicell induction or expression following minicell and/or protoplastisolation, the procedure to transform the expression constructs is thesame. To accomplish this, protein constructs were initially cloned intoE. coli MG1655 and then into the minicell producing strain of interest.Transformation events were selected prior to minicell induction. Forco-induction of protein and minicells, see the protocol for phoAexpression above. For post-minicell and/or protoplast purificationinduction experiments, following minicell purification and/or protoplastpreparation and purification, these cellular bodies were induced forprotein production in either LBD or MDT at a minicell orprotoplast/volume ratio of 1×10⁹ minicells or protoplasts 1 ml media.Media was supplemented with the appropriate inducer concentration (seeTable 6). Protein induction is sensitive to a variety of factorsincluding, but not limited to aeration and temperature, thus reactionvolume to surface area ratio is important, as is the method of shakingand temperature of induction. Therefore, each protein must be treated asrequired to optimize expression. In addition to expression parameters,protoplasted minicells are sensitive to osmotic and mechanical forces.Therefore, protoplast protein induction reactions must also contain 10%sucrose with greater volume to surface area ratios than required forintact minicells to achieve similar aeration at lower revolutions.

[1175] Using the T-PhoA as a non-limiting example, protein expressionwas performed during and following minicell isolation. To accomplishthis task, t-phoA co-expressed with minicell induction was compared tot-phoA expressed after minicell isolation. In both cases, overnightminicell-producing parental strains containing pMPX-5::t-phoA weresubcultured into LBD supplemented with the appropriate antibiotic.Cultures were grown to OD₆₀₀ 0.1 and induced for minicell productionalone or for both minicell and protein production. Both cultures wereharvested at OD₆₀₀ 1.0 and minicells produced were harvested asdescribed above. Minicells to be induced for T-phoA production followingpurification were induced by introducing 1×10⁹ purified minicells into a15 ml culture tube containing 1 ml MDT with 1 mM L-rhamnose. Minicellprotein induction was allowed to proceed for up to 14 hours and comparedto protein production obtained using the co-expression approach. Foreach approach, minicells were fractionated and analyzed for membraneassociation, total protein, and membrane association-dependent enzymaticactivity. These observations were compared to post-induction,pre-isolation parental cell/minicell (PC/MC) mixtures from theco-expressed reactions. The first observation was that co-expression ofminicell and protein induction was superior to post-minicellpurification induction (Table 18). However, although the kinetics areslower for the post-minicell purification induction protocol, the endresult is equivalent. TABLE 18 COMPARATIVE EXPRESSION: CO-EXPRESSIONVERSUS POST MINICELL PURIFICATION INDUCTION Time of induction Purifiedminicell induction ^(a) Co-expression induction ^(a) 1.0 8.0 — 2.0 —812.2 4.0 70.0 — 14.0 445.0 —

[1176] Using the co-expression induction procedure, the amount ofmembrane-associated T-PhoA was measured and compared for both parentalcells and minicells. Briefly, following co-expression induction ofT-PhoA and minicells, minicells were purified and their membranesisolated. For membrane isolation, minicells containing expressed T-PhoAwere subjected to three rounds of freeze-thaw lysis in the presence of10 μg/ml lysozyme. Following freeze-thaw cycling, the reaction wassubjected to sonication. Sonicated material was centrifuged at 6,000 rpmin a microcentrifuge for 5 min at room temperature. Supernatants weretransferred to a fresh 1.5 ml Eppendorf tube and centrifuged at 70,000rpm using a TLA-100 rotor. Following centrifugation, the pellet wasresuspended in buffer and analyzed for total T-PhoA protein (Table 19)and T-PhoA enzyme activity (Table 20). TABLE 19 MEMBRANE ASSOCIATEDT-PHOA: PARENTAL CELLS VERSUS MINICELLS Protein T-PhoA T-PhoA ProteinT-PhoA T-PhoA membrane membrane % membrane Cell type ^(a) total ^(a)total ^(b) % total associated ^(a) associated ^(b) protein totalParental cells 107.5 5.3 4.9 10.7 3.1 29.0 Minicells 4.6 0.8 17.5 1.00.5 50.0 Minicells EQ ^(b) 25.2 4.4 — 5.5 2.7 —

[1177] TABLE 20 PHOA ENZYMATIC ACTIVITY^(a) (RELATIVE UNITS): PARENTALCELLS VERSUS MINICELLS. Cell type ^(b) Unlysed Lysed, total Lysed,membrane Parent cell — 358 240 Minicell 275 265 211 Minicell EQ ^(c)1,504 1,447 1,154

[1178] These results suggest that co-expression induction of T-PhoA andminicells together results in minicells containing an equivalent amountof T-PhoA produced in both parental cells and minicells. However, thepercent of T-PhoA compared to total protein is 3.5×greater in minicellsthan in parental cells. Furthermore, of the protein made, T-PhoAconstitutes 50% of the total membrane protein in minicells, whereas itis only 29% in parental cells. It should be noted that the T-PhoAprotein associated with the membrane can be easily removed by treatmentwith mild, non-ionic detergent suggesting that the T-PhoA present in themembrane pellet is indeed associated with the membrane and not aninsoluble, co-sedimenting precipitate (data not shown). Finally, PhoA isa periplasmic enzyme that requires export to the periplasmic space forproper folding and disulfide bond formation. Both of which are requiredfor enzymatic activity. In the time course of this experiment,expression of ΔPhoA lacking a leader sequence does not demonstrateenzymatic activity. Furthermore, there is no difference between unlysedand lysed minicells containing expressed T-PhoA (Table 20) alsodemonstrating that the PhoA enzyme domain of the T-PhoA chimera must bepresent in the periplasmic space. Therefore, the T-PhoA construct mustmembrane associate and the PhoA domain must orient into the periplasmicspace for enzymatic activity. Thus, when comparing equivalent amounts ofmembrane lipid between parental cells and minicells in Table 20,membrane association-dependent T-PhoA activity is almost 5×greater thanin parental cells. Taking into account the data in Table 19 where 50% ofT-PhoA is in the membrane compared to 29% in parental cells, thedifference in T-PhoA membrane association is not sufficient to explainthe almost 5×increase in minicell activity. These observations suggestthat minicells contain a capacity to support more expressed membraneprotein than parental cells and that the protein that associates withthe membrane is more active. This activity may be simply result fromminicells allowing greater efficiency of folding and disulfide bondformation for this particular protein. However, do to the fact thatminicells do not contain chromosome, it is also possible that theoverexpression of this protein is readily finding membrane-binding sitesin the absence of chromosomally produced competitors present in parentalcells. Furthermore, overexpression of proteins often leads to increasedprotease expression. Because minicells do not contain chromosome, theseotherwise degraded surplus T-PhoA is allowed the continued opportunityto insert and properly fold in the membrane, an attribute that couldlend favor to overexpression of more complex membrane proteins.

Example 18 Exemplary Methods to Induce and Study Complex MembraneProteins

[1179] Expression of non-native (exogenous) complex membrane proteins inbacterial systems can be difficult. Using the minicell system, we areable to eliminate toxicity issues. However, issues still remain withproper translation, compartmentalization at the membrane, insertion inthe membrane and proper folding for native activity. To account forthese potential problems we have constructed a modular chimeric systemthat incorporates leader sequences and chaperone-recognized solubledomains that are native to our bacterial minicell system. In addition,we created modular constructs that overexpress the native chaperonesgroESL and trigger factor (tig). Finally, we have constructedminicell-producing strains that contain mutations that effect proteinexport and disulfide bond formation. For non-limiting examples of theseconstructs see Table 21. TABLE 21 NON-LIMITING TOOLS FOR EXOGENOUSCOMPLEX PROTEIN SYNTHESIS AND FUNCTION Residues of Tool Referencesequence Purpose SEQ ID NO pMPX-5::phoA leader — 1-48 Membrane targeting22 pMPX-5::phoA leader —  1-494 Membrane targeting 23 pMPX-5::malEleader 1 1-28 Membrane targeting 24 pMPX-5::malE leader 1  1-370Membrane targeting 25 pMPX-17 (groESL, tig) — — Chaperone 26pMPX-5::trxA::FLAG 2   2-109 ^(a)   Solubility 27

[1180] References to Table 21.

[1181] 1. Grisshammer, R., et al. 1993. Expression of a rat neurotensinreceptor in Escherichia coli. Biochem. J. 295:571-576.

[1182] 2. Tucker, J., and R. Grisshammer. 1996. Purification of a ratneurotensin receptor expressed in Escherichia coli. Biochem. J.317:891-899. TABLE 22 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE 21CONSTRUCTS SEQ ID NO.: Primer name 5′to 3′sequence 115 PhoA lead-1GTCACGGCCGAGACTTATAGTCGC 116 PhoA lead-2 GGTGTCCGGGCTTTTGTCACAGG 117PhoA lead-1-PstI CGCGGCTGCAGATGTCACGGCCGAGACTTATAGTCG C 118 PhoAlead-2-XbaI CGCGGTCTAGATTCTGGTGTCCGGGCTTTTGTCACAG G 119 PhoA completeCAGCCCCAGAGCGGCTTTCATGG 120 PhoA complete-2-XbaICGCGGTCTAGATTTCAGCCCCAGAGCGGCTTTCATG G 121 MalE lead-1CGCGGCTGCAGATGAAAATAAAAACAGGTGCACGCATCCTCGCATTATCCGCATTAACGACGATGATGTTTT CCGCCTCGGCTCTCGCCAAAATCTCTAGACGCGG122 MalE lead-2 CCGCGTCTAGAGATTTTGGCGAGAGCCGAGGCGGAAAACATCATCGTCGTTAATGCGGATAATGCGAGGATG CGTGCACCTGTTTTTTATTTTCATCTGCAGCCGCG123 MalE-1 GGTGCACGCATCCTCGCATTATCCGC 124 Ma1E-2CGGCATACCAGAAAGCGGACATCTGC 125 MalE-1-PstICGCGGCTGCAGATGAAAATAAAAACAGGTGCACGC ATCCTCGCATTATCCGC 126 MalE-2-XbaICGCGGTCTAGAACGCACGGCATACCAGAAAGCGGA CATCTGC 127 Tig-1CGCGACAGCGCGCAATAACCGTTCTCG 128 Tig-2 GCTGGTTCATCAGCTCGTTGAAAGTGG 129Tig-1-NarI GCGCCGGCGCCATACGCGACAGCGCGCAATAACCGT TCTCG 130 Tig-2-XbaIGGCGCTCTAGATTATTATTACGCCTGCTGGTTCATCA GCTCGTTGAAAGTGG 131 Gro-1GGTAGCACAATCAGATTCGCTTATGACGG 132 Gro-2 GCCGCCCATGCCACCCATGCCGCCC 133Gro-1-XbaI GCGTCTAGAGGTAGCACAATCAGATTCGCTTATGAC GG 134 Gro-2-HindIIIGGCGCAAGCTTATTATTACATCATGCCGCCCATGCC ACCCATGCCGCCC 135 TrxA-1GCGATAAAATTATTCACCTGACTGACG 136 TrxA-2 GCGTCGAGGAACTCTTTCAACTGACC 137TrxA-1-Fxa-PsfI CGCGGCTGCAGATGATCGAAGCCCGCTCTAGACTCGAGAGCGATAAAATTATTCACCTGACTGACG 138 TrxA-2-FLAG-BamHICCGCGGGATCCTTATTAATCATCATGATCTTTATAATCGCCATCATGATCTTTATAATCCTCGAGCGCCAGGTT AGCGTCGAGGAACTCTTTCAACTGACC

[1183] Oligonucleotides SEQ ID NOS.:115, 116, 117 and 118 were used toamplify the phoA leader (residues 1-49) from E. coli chromosomal DNA.Once amplified, this region was inserted into SEQ ID NO.: 6 (pMPX-5)using PstI and XbaI to create SEQ ID NO.:22.

[1184] Oligonucleotides SEQ ID NOS.:115, 117, 119 and 120 were used toamplify the complete phoA gene from E. coli chromosomal DNA. Onceamplified, this region was inserted into SEQ ID NO.: 6 (pMPX-5) usingPstI and XbaI to create SEQ ID NO.23.

[1185] Oligonucleotides SEQ ID NOS.:121 and 122 were used to constructthe malE leader (residues 1-28) sequence. Once annealed, this constructwas inserted into SEQ ID NO.: 6 (pMPX-5) using PstI and XbaI to createSEQ ID NO.:24.

[1186] Oligonucleotides SEQ ID NOS.:123, 124, 125 and 126 were used toamplify the malE expander leader (residues 1-370) from E. colichromosomal DNA. Once amplified, this region was inserted into SEQ IDNO.: 6 (pMPX-5) using PstI and XbaI to create SEQ ID NO.:25.

[1187] Oligonucleotides SEQ ID NOS.:127, 128, 129 and 130 were used toamplify the tig control and gene region from E. coli chromosomal DNA.Once amplified, this region was ligated to the groESL amplified regionbelow using XbaI prior to insertion into SEQ ID NO.: 6 (pMPX-5) usingNarI (from the tig region) and HindIII (from the groESL region) tocreate SEQ ID NO.:26.

[1188] Oligonucleotides SEQ ID NOS.:131, 132, 133 and 134 were used toamplify the groESL control and gene region from E. coli chromosomal DNA.Once amplified, this region was ligated to the tig amplified regionabove using XbaI prior to insertion into SEQ ID NO.: 6 (pMPX-5) usingNarI (from the tig region) and HindIII (from the groESL region) tocreate SEQ ID NO.:26.

[1189] Oligonucleotides SEQ ID NOS.:135, 136, 137 and 138 were used toamplify trxA (residues 2-109) from E. coli chromosomal DNA and insertFLAG and Factor Xa sequences. Once amplified, this region was insertedinto SEQ ID NO.: 6 (pMPX-5) using PstI and BamHI to create SEQ IDNO.:27.

[1190] By way of non-limiting example, the pMPX-5::phoA leader (residues1-48), pMPX-5::phoA leader (residues 1-494), pMPX-5::malE leader(residues 1-28), and pMPX-5::malE leader (residues 1-370) constructs aredesigned to direct expressed exogenous membrane proteins to the minicellcytoplasmic membrane. In addition to these constructs, By way ofnon-limiting example, mutations in E. coli genes secA and secY,specifically mutation prlA4 (Strader, J., et al. 1986. Kinetic analysisof lamB mutants suggests the signal sequence plays multiple roles inprotein export. J. Biol. Chem. 261:15075-15080), permit promiscuoustargeting to the membrane. These mutations, like the above constructsare integrated into the minicell expression system. To complement thesemutations, the chaperone complex groESL and trigger factor have alsobeen incorporated into the expression system. By way of non-limitingexample, pMPX-5::trxA::FLAG will be used to create a carboxy-terminalfusion to the protein of interest to increase the membrane insertionefficiency of the membrane protein of interest (Tucker, J., and R.Grisshammer. 1996. Purification of a rat neurotensin receptor expressedin Escherichia coli. Biochem. J. 317:891-899). Also By way ofnon-limiting example, pMPX-5::FLAG::toxR and pMPX-5::FLAG::λcIconstructs will be prepared to create a carboxy-terminal fusion to theprotein of interest for use in a reporter-based assay forprotein-protein interactions. By way of non-limiting example, theprotein of interest for this system is a GPCR. Also By way ofnon-limiting example, this GPCR may be the neurotensin receptor from rat(Grisshammer, R., et al. 1993. Expression of a rat neurotensin receptorin Escherichia coli. Biochem. J. 295:571-576.), or the β2 adrenergicreceptor from humans (Freissmuth, M., et al. 1991. Expression of twoβ-adrenergic receptors in Escherichia coli: functional interaction withtwo forms of the stimulatory G protein. Proc. Natl. Acad. Sci.88:8548-8552). Insertion of a GPCR into one of these reporter constructscreates a carboxy-terminal fusion between the GPCR of interest and theDNA-binding regulatory domain of the ToxR positive activator, the λcIrepressor, or the AraC positive activator. To complete this reportersystem, By way of non-limiting example pMPX-5::(X)::toxR orpMPX-5::(X)::λcI will be used to create a carboxy-terminal fusion to theprotein of interest for use in a reporter-based assay forprotein-protein interactions, where (X) may be any protein or moleculeinvolved in an intermolecular or intramolecular interaction. By way ofnon-limiting example, this molecule of interest may be a G-protein. ThisG-protein may be the Gα_(i1)-protein from rat (Grisshammer, R., and E.Hermans. 2001. Functional coupling with Gαq and Gαi1 protein subunitspromotes high-affinity agonist binding to the neurotensin receptor NTS-1expressed in Escherichia coli. FEBS Lett. 493:101-105), or theGs_(α)-protein from human (Freissmuth, M., et al. 1991. Expression oftwo β-adrenergic receptors in Escherichia coli: functional interactionwith two forms of the stimulatory G protein. Proc. Natl. Acad. Sci.88:8548-8552). Like the GPCR, insertion of a G-protein into one of thesereporter constructs creates a carboxy-terminal fusion between theG-protein of interest and the DNA-binding regulatory domain of the ToxRpositive activator, the λcI repressor, or other regulatory protein.Finally, these plasmid constructs contain the DNA-binding domain of eachregulator; the ctx regulatory region from Vibrio cholerae (Russ, W. P.,and D. M. Engelman. 1999. TOXCAT: a measure of transmembrane helixassociation in a biological membrane. 96:863-868), or the PR1OR1regionof bacteriophage lambda (Hu, J. C., et al. 1990. Sequence requirementsfor coiled-coils: analysis with lambda repressor-GCN4 leucine zipperfusions. Science. 250:1400-1403), respectively. By way of non-limitingexample, each binding domain is coupled to a reporter sequence encodingluciferase (Dunlap, P. V., and E. P. Greenberg. 1988. Control of Vibriofischeri lux gene transcription by a cyclic AMP receptor protein-luxRprotein regulatory circuit. J. Bacteriol. 170:4040-4046), greenfluorescent protein (GFP) (Yang, T. T., et al. 1996. Dual colormicroscopic imagery of cells expressing the green fluorescent proteinand a red-shifted variant. Gene. 173:19-23; Matthysse, A. G., et al.1996. Construction of GFP vectors for use in gram-negative bacteriaother than Escherichia coli. FEMS Microbiol. Lett. 145:87-94), or otherreporter. Co-expression of these GPCR and G-protein chimeras will createa system measuring the interaction between a GPCR and G-protein withinan intact minicell. This system is designed to be used as a positive ornegative read-out assay and may be used to detect loss or gain of GPCRfunction. Although the GPCR-G-protein interaction is provided as anexample, this modular system may be employed with any soluble ormembrane protein system measuring protein-protein or otherintermolecular interaction.

Example 19 Exemplary Methods for Gene Transfer Using Minicells orMinicell Protoplasts

[1191] Included in the design of the invention is the use of minicellsto transfer genetic information to a recipient cell. By way ofnon-limiting example, this gene transfer may occur between a minicelland a mammalian cell in vitro, or in vivo, and this gene transfer mayoccur through cell-specific interactions, through general interactions,or a combination of each. To accomplish this task three basic constructswere created. Each of these constructs is created in pMPX-6 whichcontains a CMV promotor controlling the synthesis of GFP. The plasmidpMPX-6 was constructed by cloning the araC through the multiple cloningsite of pBAD24 into pEGFP (Clontech). This construct provided abacterial regulator as well as a method to monitor the success of genetransfer using GFP expression form the CMV promotor. In design, theprotein expressed using the bacterial promotor will drive the cell-cellinteraction, while the successful transfer of DNA from the minicell tothe recipient cell will initiate the production of GFP. By way ofnon-limiting example, proteins that will drive the cell-cell interactionmay be the invasin protein from Yersinia pseudotuberculosis, whichstimulates β1 integrin-dependent endocytic events. To properly displaythe invasin protein on the surface of minicells, the domain of invasinthat stimulates these events (residues 490-986) (Dersch, P., and R. R.Isberg. 1999. A region of the Yersinia pseudotuberculosis invasinprotein enhances integrin-mediated uptake into mammalian cells andpromotes self-association. EMBO J. 18:1199-1213) was fused to thetransmembrane domain of ToxR. Expression of this construct from pMPX-6will display T-Inv on the, surface of the minicell and stimulateendocytosis with any cell displaying a β1 integrin. Thus, T-Inv displaywill provide a general mechanism of gene transfer from minicells. Toprovide specificity, By way of non-limiting example, the ligand portionof epidermal growth factor (EGF) may be fused to the transmembranedomain of ToxR, thus creating a protein that will interact with cellsdisplaying the EGF receptor (EGFR). Likewise, tumor nucrosis factor(TNF) may also serve this purpose by stimulating cell-cell interactionsbetween minicells displaying TNF and cells displaying TNF receptor(TNFR). Although EGF-EGFR and TNF-TNFR interactions may stimulatecell-cell fusion between minicells and recipient cells, or minicelluptake, this alone may not be sufficient to efficiently transfer geneticinformation from minicells. Therefore, a genetic approach to increasingthe cell-cell genetic transfer may be the development of a geneticswitch that senses the specificity interaction, e.g. EGF-EGFRinteraction, and turns on the production of a second gene product, e.g.invasin, that stimulates the endocytic event. By way of non-limitingexample, this genetic switch may be similar to the GPCR-G-proteininteraction reporter system above, in that an extracellular eventstimulates the dimerization of a transcriptional active regulator, thusturning on the production of invasin or invasin-like protein. In eitherapproach, the display system to stimulate transfer of geneticinformation from minicells to recipient cells may also be applicable tothe transfer of substances other than genetic information, e.g.pre-synthesized therapeutic drugs.

[1192] To test this targeting methodology, different pMPX-6 constructscontaining each of these general or specific cell-cell interactionproteins will be transformed into a minicell producing strain and eitherby co-expression induction of minicells, by post-minicell purificationinduction, or by post-protoplasting induction, minicells displaying thetargeting protein of interest will be produced. When using theco-expression induction and post-minicell purification induction of thetargeting protein approaches, it is necessary to protoplast the purifiedminicells after protein induction. Once the targeting protein has beendisplayed on the surface of a minicell protoplast, these protoplasts areready to be exposed to target cells. For preliminary experiments theseinteractions will be monitored using cell culture of Cos cells incomparison to lipofectamine (Invitrogen), electroporation, and othertransfection techniques. Initial experiments will expose protoplastsdisplaying T-Inv to Cos cells and compare the transfection efficiency toprotoplast containing pMPX-6::t-inv in the absence of t-inv expression,naked pMPX-6::t-inv alone, and naked pMPX-6::t-inv with lipofectamine.Each of these events will be monitored using fluorescent microscopyand/or flow cytometry. From these results the specific targetingapparatus proteins will be tested. Using A-431 (display EGFR) and K-562(no EGFR) cell lines, the pMPX-6::t-egf constructs will be tested. Usingthe same approaches as for the t-inv study, the level of transfectionbetween A-431 and K-562 cell lines will be measured and compared tothose achieved using lipofectamine. Similarly, the ability of TNF tostimulate gene transfer will be studied using L-929 cells. In all cases,the ability of these general and specific targeting protein constructswill be compared to standard transfection techniques. Upon positiveresults, these methodologies will be tested on difficult to transfectcell lines, e.g. adult cardiomyocytes. The basis of these results willcreate a foundation for which applications into in vivo gene transfermay occur.

Example 20 Additional and Optimized Methods for Genetic Expression

[1193] Expression in minicells may occur following purification ofminicells and/or minicell protoplasts from parental cells and LPSconstituents, respectively. However, for some applications it ispreferred to co-express proteins of interest with minicell induction.For these approaches, one may use the protocol described in Example 13for expression of the phoa constructs. Either of these approaches may beaccomplished using one or more of the following expression constructs(Table 23) and/or optimized expression constructs (Table 25).

[1194] Expression plasmid pCGV1 contains a temperature sensitive lambdacI repressor (cI857) and both lambda PR and PL promoters (Guzman, C. A.,et al. 1994. A novel Escherichia coli expression-export vectorcontaining alkaline phosphatase as an insertional inactivation screeningsystem. Gene. 148:171-172) with an atpE initiation region (Schauder, B.,et al. 1987. Inducible expression vectors incorporating the Escherichiacoli atpE translational initiation region. Gene. 52:279-283). Includedin the design of the invention is the modification of this expressionvector to best align the required Shine-Delgarno ribosomal binding sitewith cloning sites. In addition, the pCGVI expression vector wasmodified to incorporate a stem-loop structure at the 3-prime end of thetranscript in order to provide a strong transcriptional stop sequence(Table 23).

[1195] Expression plasmid pCL478 contains a temperature sensitive lambdacI repressor (cI857) and both lambda PR and PL promoters (Love, C. A.,et al. 1996. Stable high-copy bacteriophage promoter vectors foroverproduction of proteins in Escherichia coli. Gene. 176:49-53).Included in the design of the invention is the modification of thisexpression vector to best align the required Shine-Delgarno ribosomalbinding site with cloning sites. In addition, the pCL478 expressionvector was modified to incorporate a stem-loop structure at the 3-primeend of the transcript in order to provide a strong transcriptional stopsequence (Table 23). TABLE 23 LAMBDA CI857 EXPRESSION VECTORMODIFICATIONS New Parent Region Plasmid plasmid removed Region added^(a) SEQ ID NO pMPX-84 pCGV1 NdeI - BamHI NdeI, SD - PstI, XbaI, KpnI,Stem-loop, BamHI 139 pMPX-85 pCGV1 NdeI - BamHI NdeI, SD - SalI, XbaI,KpnI, Stem-loop, BamHI 140 pMPX-86 pCL478 BamHI - XhoI BamHI, SD - PstI,XbaI, KpnI, Stem-loop, XhoI 141 pMPX-87 pCL478 BamHI - XhoI BamHI, SD -SalI, XbaI, KpnI, Stem-loop, XhoI 142

[1196] TABLE 24 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE 23 SEQ ID NOPrimer name 5′to 3′sequence 143 CGV1-1-SalITATGTAAGGAGGTTGTCGACCGGCTCAGTCTAGAGGTACCCGCCCTCA TCCGAAAGGGCGTATTG 144CGV 1-2-SalI GATCCAATACGCCCTTTCGGATGAGGGCGGGTACCTCTAGACTGAGCCCGTCGACAACCTCCTTACA 145 CGV1-1-PstITATGTAAGGAGGTTCTGCAGCGGCTCAGTCTAGAGGTACCCGCCCTCA TCCGAAAGGGCGTATTG 146CGV1-2-PstI GATCCAATACGCCCTTTCGGATGAGGGCGGGTACCTCTAGACTGAGCCGCTGCAGAACCTCCTTACA 147 CL478-1-SalIGATCCTAAGGAGGTTGTCGACCGGCTCAGTCTAGAGGTACCCGCCCTC ATCCGAAAGGGCGTATTC 148CL478-2-SalI TCGAGAATACGCCCTTTCGGATGAGGGCGGGTACCTCTAGACTGAGCCGGTCGACAACCTCCTTAG 149 CL478-1-PstIGATCCTAAGGAGGTTCTGCAGCGGCTCAGTCTAGAGGTACCCGCCCTC ATCCGAAAGGGCGTATTC 150CL478-2-PstI TCGAGAATACGCCCTTTCGGATGAGGGCGGGTACCTCTAGACTGAGCCGCTGCAGAACCTCCTTAG

[1197] Oligonucleoides SEQ ID NOS.: 143 and 144 were annealed to eachother to generate a DNA molecule with a 5′ overhang at both ends. Theoverhangs are designed so that the DNA can be directly cloned into pCGVIcut with NdeI (5′ overhang is TA) and BamHI (5′ overhang is GATC).Insertion of the annealed DNA into pCGVI creates SEQ ID NO.: 139,pMPX-84.

[1198] Oligonucleoides SEQ ID NOS.: 145 and 146 were annealed to eachother to generate a DNA molecule with a 5′ overhang at both ends. Theoverhangs are designed so that the DNA can be directly cloned into pCGVIcut with NdeI (5′ overhang is TA) and BamHI (5′ overhang is GATC).Insertion of the annealed DNA into pCGVI creates SEQ ID NO.: 140,pMPX-85.

[1199] Oligonucleoides SEQ ID NOS.: 147 and 148 were annealed to eachother to generate a DNA molecule with a 5′ overhang at both ends. Theoverhangs are designed so that the DNA can be directly cloned intopCL478 cut with BamHI (5′ overlap is GATC) and XhoI (overhang is TCGA).Insertion of the annealed DNA into pCL578 cut with BamHI and XhoIcreates SEQ ID NO.: 141, pMPX-86.

[1200] Oligonucleoides SEQ ID NOS.: 149 and 150 were annealed to wereannealed to each other to generate a DNA molecule with a 5′ overhang atboth ends. The overhangs are designed so that the DNA can be directlycloned into pCL578 cut with BamHI (5′ overlap is GATC) and XhoI(overhang is TCGA). Insertion of the annealed DNA into pCL478 cut withBamHI and XhoI creates SEQ ID NO.: 142, pMPX-87.

[1201] The optimized expression constructs in Table 25 were created fromSEQ ID NOS.: 6, 11, and 12 (see Table 13). Modifications were made tooptimize the alignment of the SalI or PstI cloning sites with theShine-Delgarno ribosome-binding site. In addition, stem-looptranscriptional termination sequences were added on the 3′ end of thecloning region. TABLE 25 EXPRESSION CONSTRUCTS Plasmid Regulatoryelement(s) inducer Plasmid SEQ ID NO.: pMPX-67 RhaRS Rhamnose PUC-18 151pMPX-72 RhaRS Rhamnose PUC-18 152 pMPX-66 AraC Arabinose PUC-18 153pMPX-71 AraC Arabinose PUC-18 154 pMPX-68 MelR Melibiose PUC-18 155

[1202] TABLE 26 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE 25 CONSTRUCTSSEQ ID NO.: Primer name 5′to 3′sequence 69 Rha-1GCGAATTGAGATGACGCCACTGGC 156 Rha-SD GCAGAACCTCCTGAATTTCATTACGACC 71Rha-1-HindIII CGGCGAAGCTTAATTAATCTTTCTGCGAATTGAGATGACGC CACTGGC 157Rha-SD SalI CCGCGGGTACCAATACGCCCTTTCGGATGAGGGCGCGGGG KpnIATCCTCTAGAGTCGACGTCGACAACCTCCTGAATTTCATTA CGACC 158 Rha-SD KpnICCGCGGGTACCAATACGCCCTTTCGGATGAGGGCGCGGGG KpnIATCCTCTAGAGTCGACCTGCAGAACCTCCTGAATTTCATTA CGACC 81 Ara-1CAAGCCGTCAATTGTCTGATTCG 159 Ara-SDCTGCAGGGCCTCCTGCTAGCCCAAAAAAACGGGTATGG 83 Ara-1-HindIIIGCGCCAAGCTTCAAGCCGTCAATTGTCTGATTCG 160 Ara-SD SalICCGCGGGTACCAATACGCCCTTTCGGATGAGGGCGCGGGG KpnIATCCTCTAGAGTCGACGTCGACGGCCTCCTGCTAGCCCAAA AAAACGGGTATGG 161 Ara-SD PstICCGCGGGTACCAATACGCCCTTTCGGATGAGGGCGCGGGG KpnIATCCTCTAGAGTCGACCTGCAGGGCCTCCTGCTAGCCCAAA AAAACGGGTATGG 77 Mel-1CGTCTTTAGCCGGGAAACG 162 Mel-SD CCTCCTGGCTTGCTTGAATAACTTCATCATGG 79Mel-1-HindIII GCCGCAAGCTTCGTCTTTAGCCGGGAAACG 163 Mel-SD-SaliCCGCGGGTACCAATACGCCCTTTCGGATGAGGGCGCGGGG KpnIATCCTCTAGAGTCGACCCCCTCCTGGCTTGCTTGAATAACT TCATCATGGC

[1203] Oligonucleotides SEQ ID NOS.: 69, 156, 72, and 157 were used toamplify the rhaRS genes and their divergent control region from the E.coli chromosome and insertion of an optimized SalI-Shine-Delgarnoribosome-binding alignment and a stem-loop transcriptional terminationsequence. Once amplified, this region was inserted into pUC18 usingHindIII and KpnI to create pMPX67, SEQ ID NO.: 151.

[1204] Oligonucleotides SEQ ID NOS.: 69, 156, 72, and 158 were used toamplify the rhaRS genes and their divergent control region from the E.coli chromosome and insertion of an optimized PstI-Shine-Delgamoribosome-binding alignment and a stem-loop transcriptional terminationsequence. Once amplified, this region was inserted into pUC18 usingHindIII and KpnI to create, pMPX-72, SEQ ID NO.: 152.

[1205] Oligonucleotides SEQ ID NOS.: 81, 159, 81, 160 were used toamplify the araC genes and their divergent control region from the E.coli chromosome and insertion of an optimized SalI-Shine-Delgarnoribosome-binding alignment and a stem-loop transcriptional terminationsequence. Once amplified, this region was inserted into pUC18 usingHindIII and KpnI to create, pMPX-66, SEQ ID NO.: 153.

[1206] Oligonucleotides SEQ ID NOS.: 81, 159, 81, 161 were used toamplify the araC genes and their divergent control region from the E.coli chromosome and insertion of an optimized PstI-Shine-Delgarnoribosome-binding alignment and a stem-loop transcriptional terminationsequence. Once amplified, this region was inserted into pUC18 usingHindIII and KpnI to create pMPX-71, SEQ ID NO.: 154.

[1207] Oligonucleotides SEQ ID NOS.: 77, 162, 79, 163 were used toamplify the melR genes and their divergent control region from the E.coli chromosome and insertion of an optimized SalI-Shine-Delgarnoribosome-binding alignment and a stem-loop transcriptional terminationsequence. Once amplified, this region was inserted into pUC18 usingHindIII and KpnI to create, pMPX-68, SEQ ID NO.: 155.

Example 21 Optimization of Rat Neurotensin Receptor (NTR) Expression

[1208] Expression of specific GPCR proteins in minicells may requirechimeric domain fusions to stabilize the expressed protein and/or directthe synthesized protein to the membrane. The NTR protein from rat wascloned into several chimeric combinations to assist in NTR expressionand membrane association (Grisshammer, R., et al. 1993. Expression of arat neurotensin receptor in Escherichia coli. Biochem. J. 295:571-576;Tucker, J., and Grisshammer, R. 1996. Purification of a rat neurotensinreceptor expressed in Escherichia coli. Biochem. J. 317:891-899).Methods for construction are shown the Tables below. TABLE 27NEUROTENSIN RECEPTOR EXPRESSION FACILITATING CONSTRUCTS Protein ^(a)Construct ^(b) SEQ ID NO MalE(L) SalI-MalE (1-370)-Factor Xa-NTRhomology 164 NTR Factor Xa-NTR (43-424)-NotI-FLAG-KpnI 165 MalE(L)-NTRSalI-MalE(1-370)-Factor Xa-NTR(43-424)-NotI-FLAG-KpnI 166 MalE(S)-NTRSalI-MalE(1-28)-Factor Xa-NTR(43-424)-NotI-FLAG-KpnI 167 TrxANotI-TrxA(2-109)-NotI 168 MalE(L)-NTR-TrxA SalI-MalE(1-370)-FactorXa-NTR(43-424)-NotI-TrxA(2-109)- 169 FLAG-KpnI MalE(S)-NTR-TrxASalI-MalE(1-28)-Factor Xa-NTR(43-424)-NotI-TrxA(2-109)-FLAG- 170 KpnI

[1209] TABLE 28 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE 27 SEQ IDNO.: Primer name 5′to 3′sequence 171 MalE-1 GGTGCACGCATCCTCGCATTATCCGC172 MalE-2 CGCACGGCATACCAGAAAGCGGACATCTGCG 173 MalE-1-SalICCGCGGTCGACATGAAAATAAAAACAGGTGCACGCATCCTCGC 174 MalE-2-XaNTRGCCGTGTCGGATTCCGAGGTGCGGCCTTCGATACGCACGGCAT ACCAAGAAAGCGGGATGTTCGGC 175NTR-1 CCTCGGAATCCGACACGGCAGGGC 176 NTR-2 GTACAGGGTCTCCCGGGTGGCGCTGG 177NTR-1 Xa CCGCGATCGAAGGCCGCACCTCGGAATCCGACACGGCAGGGCC 178 NTR-2-FlagGGCGCGGTACCTTTGTCATCGTCATCTTTATAATCTGCGGCCGCGTACAGGGTCTCCCGGGTGGCGCTGGTGG 179 NTR-2-Stop KpnIGCGGCGGTACCTTATATTTGTCATCGTCATCTTTATAATCTGC GGCCGCG 180 NTR-1-Xa LeadCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCAAAATCATCGAAGGCCGCACCTCGGAATCCGACACGGC 181 NTR-2-Lead2 SalICCGCGGTCGACATGAAAATAAAAACAGGTGCACGCATCCTCGCATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGC 182 TrxA-1CCGCGAGCGATAAAATTATTCACCTGACTGACG 183 TrxA-2GCCCGCCAGGTTAGCGTCGAGGAACTCTTTCAACTGACC 184 TrxA-1-NotIGCGGCCGCAAGCGATAAAATTATTCACCTGACTGACG 185 TrxA-2-NotIGGCGCTGCGGCCGCATCATCATGATCTTTATAATCGCC

[1210] Oligonucleotides SEQ ID NOS.: 171, 172, 173 and 174 were used toamplify malE residues 1-370 from the E. coli chromosome to create SEQ IDNO.: 164. Using overlap PCR with the extended NTR homology, a chimerictranslational fusion was made between MalE (1-370) and NTR residues43-424 (SEQ ID NO.: 165) to create a SEQ ID NO.: 166. SEQ ID NO.: 166was cloned into plasmids pMPX-85, pMPX-87, pMPX-66 and pMPX-67(respectively, SEQ ID NOS.: 140, 142, 151 and 153) using SalI and KpnI.

[1211] Three-step PCR with oligonucleotides, SEQ ID NOS.: 175 and 176 asprimers was used to amplify NTR residues 43-424 from rat brain cDNA. SEQID NOS.: 177 and 178 were then used with the NTR (43-424) template toadd factor Xa and FLAG sequence. Finally, SEQ ID NOS.: 177 and 179 wereused to add a KpnI site to create SEQ ID NO.: 165. Using overlap PCRwith malE(1-370) containing extended NTR homology, a chimerictranslational fusion was made between NTR (43-424) and MalE (1-370) (SEQID NO.: 164) to create a SEQ ID NO.: 166. SEQ ID NO.: 166 was clonedinto SEQ ID NOS.: 140, 142, 151 and 153 using SalI and KpnI.

[1212] Using three-step PCR oligonucleotides SEQ ID NOS.: 175 and 176were first used to amplify NTR residues 43-424 from rat brain cDNA. SEQID NOS.: 178 and 180 were then used with the NTR (43-424) template toadd factor Xa and FLAG sequence. Finally, SEQ ID NOS.: 179 and 181 wereused to add KpnI to create SEQ ID NO.: 167. SEQ ID NO.: 167 was clonedinto SEQ ID NOS.: 140, 142, 151 and 153 using SalI and KpnI.

[1213] Oligonucleotides SEQ ID NOS.: 182, 183, 184 and 185 were used toamplify TrxA residues 2-109 from the E. coli chromosome to create SEQ IDNO.: 168. Using NotI, TrxA residues 2-109 was cloned into SEQ ID NOS.:166 and 167 to create SEQ ID NOS.: 169 and 170, respectively. SEQ IDNO.: 169 and 170 were cloned into SEQ ID NOS.: 140, 142, 151 and 153using SalI and KpnI.

Example 22 Methods for Functional GPCR Assay

[1214] Functional G-protein-coupled receptor (GPCR) binding assays inminicells requires expression of a GPCR of interest into the minicellmembrane bilayer and cytoplasmic expression of the required G-protein.For these purposes, constructs were created to co-express both a GPCRand a G-protein. To regulate the ratio of GPCR to G-protein,transcriptional fusions were created. In these constructs, the GPCR andG-protein are co-transcribed as a bi-cistronic mRNA. To measure theGPCR-G-protein interaction in the intact minicell, each protein wascreated as a chimera with a transactivation domain. For these studiesthe N-terminal DNA-binding, activation domain of the ToxR protein fromV. cholerae was fused to the C-terminus of both the GPCR and G-protein.Finally, to measure the interaction (GPCR-G-protein interaction, theToxR-activated ctx promoter region was cloned in front of lacZ.Dimerization of the ToxR DNA-binding region will bind and activate thectx promoter. In this construct, heterodimerization of the GPCR andG-protein will promote dimerization of ToxR that will be monitored byLacZ expression. Details of these constructs are shown in Table 29.TABLE 29 FUNCTIONAL HUMAN GPCR CONSTRUCTS Protein ^(a,b) Construct^(a,b) SEQ ID NO.: β2AR SalI-β2AR-PstI, XhoI 186 GS1α XhoI-GS1α-XbaI 187β2AR-GS1α fusion SalI-β2AR-PstI, XhoI-GS1α-XbaI 188 β2AR-stopSalI-β2AR-PstI-Stop-SD-XhoI 189 β2AR-stop-GS1αSalI-β2AR-PstI-Stop-SD-XhoI-GS1α-XbaI 190 ToxR ClaI-ToxR-XbaI 191 GS1αXhoI-GS1α-ClaI 192 GS2α XhoI-GS2α-ClaI 193 Gαq XhoI-Gqα-ClaI 194 GiαXhoI-Giα-ClaI 195 Gα12/13 XhoI-Gα12/13-ClaI 196 GS1α-ToxRXhoI-GS1α-ClaI-ToxR-XbaI 197 GS2α-ToxR XhoI-GS2α-ClaI-ToxR-XbaI 198Gαq-ToxR XhoI-Gαq-ClaI-ToxR-XbaI 199 Giα-ToxR XhoI-Giα-ClaI-ToxR-XbaI200 Gα12/13-ToxR XhoI-Gα12/13-ClaI-ToxR-XbaI 201 ToxR PstI-ToxR-XhoI 202β2AR SalI-β2AR-PstI 203 β2AR-ToxR SalI-β2AR-PstI-ToxR-Stop-SD-XhoI 204β2AR-ToxR-stop- SalI-β2AR-PstI-ToxR-Stop-SD-XhoI-GS1α-ClaI-ToxR-XbaI 205GS1α-ToxR Pctx XbaI-Pctx-lacZ homology 206 lacZ Pctx homology-lacZ-XbaI207 Pctx::lacZ XbaI-Pctx-lacZ-XbaI 208

[1215] TABLE 30 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE 29. SEQ IDNO: Primer name 5′to 3′ sequence 209 β2AR-1 GGGGCAACCCGGGAACGGCAGCGCC210 β2AR-2 GCAGTGAGTCATTTGTACTACAATTCCTCC 211 β2AR-1-SalICGCGGTCGACATGGGGCAACCCGGGAACGGCAGCGCC 212 β2AR-2-Link-XhoIGGCTCGAGCTGCAGGTTGGTGACCGTCTGGCCACGCTC TAGCAGTGAGTCATTTGTACTACAATTCC 213GS1α-1 GGGCTGCCTCGGGAACAGTAAGACCGAGG 214 GS1α-2GAGCAGCTCGTACTGACGAAGGTGCATGC 215 GS1α-1-XhoIGGAGGCCCTCGAGATGGGCTGCCTCGGGAACAGTAAG ACCGAGG 216 GS1α-2-XbaICCTCTAGATTATTATCGATGAGCAGCTCGTACTGACGA AGGTGCATGC 217 GS1α-2-ClaICCATCGATGAGCAGCTCGTACTGACGAAGGTGCATGC 218 Gα12-1CCGGGGTGGTGCGGACCCTCAGCCGC 219 Gα12-2 CTGCAGCATGATGTCCTTCAGGTTCTCC 220Gα12-1-XhoI GCGGGCTCGAGATGTCCGGGGTGGTGCGGACCCTCAGC CGC 221 Gα12-2-ClaIGCGCCATCGATCTGCAGCATGATGTCCTTCAGGTTCTCC 222 Gαq-1GACTCTGGAGTCCATCATGGCGTGCTGC 223 Gαq-2 CCAGATTGTACTCCTTCAGGTTCAACTGG 224Gαq-1-XhoI ATGACTCTGGAGTCCATCATGGCGTGCTGC 225 Gαq-2-ClaIGCGCCATCGATGACCAGATTGTACTCCTTCAGGTTCAACTGG 226 Giα-1GGGCTGCACCGTGAGCGCCGAGGACAAGG 227 Giα-2 CCTTCAGGTTGTTCTTGATGATGACATCGG228 Giα-1-XhoI ATGGGCTGCACCGTGAGCGCCGAGGACAAGG 229 Giα-2-ClaIGCGCCATCGATGAAGAGGCCGCAGTCCTTCAGGTTGTTCTTGA TGATGACATCGG 230 GS2α-1GGGCTGCCTCGGGAACAGTAAGACCGAGG 231 GS2α-2 GAGCAGCTCGTACTGACGAAGGTGCATGC232 GS2α-1-XhoI ATGGGCTGCCTCGGGAACAGTAAGACCGAGG 233 GS2α-2-ClaIGCGCCATCGATGAGCAGCTCGTACTGACGAAGGTGCATGC 234 β2AR-2-Link-Stop-XhoIGGCTCGAGGGCCTCCTTGATTATTACTCGAGGGCCTCCTTGATTATTACTGCAGGTTGGTGACCGTCTGGCCACGC TCTAGCAGTGAGTCATTTGTACTACAATTCC235 β2AR-2-Link CCCTGCAGGTTGGTGACCGTCTGGCCACGCTCTAGCAGTGAGTCATTTGTACTACAATTCC 236 Tox (5-141)-1BGGACACAACTCAAAAGAGATATCGATGAGTCATATTGG 237 Tox (5-141)-2GAGATGTCATGAGCAGCTTCGTTTTCGCG 238 Tox (5-141)-1-LinkGCGTGGCCAGACGGTCACCAACCTGCAGGGACACAAC TCAAAAGAGATATCG 239 Tox(5-141)-2-XhoI CGGGGATCCTCTAGATTATTAAGAGATGTCATGAGCAG CTTCGTTTTCGCG 240Ctx-1 GGCTGTGGGTAGAAGTGAAACGGGGTTTTACCG 241 Ctx-2CTTTACCATATAATGCTCCCTTTGTTTAACAG 242 Ctx-2-XbaICGCGGTCTAGAGGCTGTGGGTAGAAGTGAAACGGGGT TTACCG 243 Ctx-2-LacZCGACGGCCAGTGAATCCGTAATCATGGTCTTTACCATA TAATGCTCCCTTTGTTTAACAG 244 LacZ-1CCATGATTACGGATTCACTGGCCGTCG 245 LacZ-2 CCAGACCAACTGGTAATGGTAGCGACC 246LacZ-1-Ctx GGTAAAGACCATGATTACGGATTCACTGGCCGTCG 247 LacZ-2-XbaIGCGCCTCTAGAAATACGCCCTTTCGGATGAGGGCGTTATTATTTTTGACACCAGACCAACTGGTAATGGTAGCG ACC

[1216] Oligonucleotides SEQ ID NOS.: 209, 210, 211 and 212 were used toamplify human β2AR from human cDNA to create SEQ ID NO.: 186. Using SalIand XhoI a translational fusion was made between β2AR and human GS1α(SEQ ID NO.: 187) to create a SEQ ID NO.: 188. SEQ ID NO.: 188 wascloned into SEQ ID NOS.: 140, 142, 151 and 153 using SalI and XbaI.

[1217] Oligonucleotides SEQ ID NOS.: 213, 214, 215 and 216 were used toamplify human GS1α from human cDNA to create SEQ ID NO.: 187. Using XhoIand XbaI a translational fusion was made between GS1α and human β2AR(SEQ ID NO.: 186) create SEQ ID NO.: 188. SEQ ID NO.: 188 was clonedinto SEQ ID NOS.: 140, 142, 151 and 153 using SalI and XbaI.

[1218] Oligonucleotides SEQ ID NOS.: 213, 214, 215 and 217 were used toamplify human GS1α from human cDNA to create SEQ ID NO.: 192. Using XhoIand XbaI a translational fusion was made with ToxR residues 5-141 fromVibrio cholerae (SEQ ID NO.: 191) to create SEQ ID NO.: 197. To be usedto create a transcriptional fusion with β2AR-ToxR chimeras as shown inSEQ ID NO.: 205 and future GPCR-ToxR chimeras.

[1219] Oligonucleotides SEQ ID NOS.: 218, 219, 220 and 221 were used toamplify human Gα12/13 from human cDNA to create SEQ ID NO.: 196. UsingXhoI and XbaI a translational fusion was made ToxR residues 5-141 fromVibrio cholerae (SEQ ID NO.: 191) to create SEQ ID NO.: 201. To be usedto create future transcriptional fusions with GPCR-ToxR chimeras asshown in SEQ ID NO.: 205.

[1220] Oligonucleotides SEQ ID NOS.: 222, 223, 224 and 225 were used toamplify human Gαq from human cDNA to create SEQ ID NO.: 194. Using XhoIand XbaI a translational fusion was made with ToxR residues 5-141 fromVibrio cholerae (SEQ ID NO.: 191) to create SEQ ID NO.: 199. To be usedto create future transcriptional fusions with GPCR-ToxR chimeras asshown in SEQ ID NO.: 205.

[1221] Oligonucleotides SEQ ID NOS.: 226, 227, 228 and 229 were used toamplify human Giα from human cDNA to create SEQ ID NO.: 195. Using XhoIand XbaI a translational fusion was made with ToxR residues 5-141 fromVibrio cholerae (SEQ ID NO.: 191) to create SEQ ID NO.: 200. To be usedto create future transcriptional fusions with GPCR-ToxR chimeras asshown in SEQ ID NO.: 205.

[1222] Oligonucleotides SEQ ID NOS.: 230, 231, 232 and 233 were used toamplify human GS2α from human cDNA to create SEQ ID NO.: 193. Using XhoIand XbaI a translational fusion was made with ToxR residues 5-141 fromVibrio cholerae (SEQ ID NO.: 191) to create SEQ ID NO.: 198. To be usedto create future transcriptional fusions with GPCR-ToxR chimeras asshown in SEQ ID NO.: 205.

[1223] Oligonucleotides SEQ ID NOS.: 209, 210, 211 and 234 were used toamplify human β2AR from human cDNA to create SEQ ID NO.: 189. Using SalIand XhoI a transcriptional fusion was made between β2AR and human GS1α(SEQ ID NO.: 187) to create a SEQ ID NO.: 190. SEQ ID NO.: 190 wascloned into SEQ ID NOS.: 140, 142, 151 and 153 using SalI and XbaI.

[1224] Oligonucleotides SEQ ID NOS.: 236, 237, 238 and 239 were used toamplify bases coinciding with ToxR residues 5-141 from Vibrio Choleraeto create SEQ ID NO.: 202. Using PstI and XhoI a translational fusionwas made between ToxR and human β2AR (SEQ ID NO.: 203) to create SEQ IDNO.: 204.

[1225] Oligonucleotides SEQ ID NOS.: 209, 210, 211 and 235 were used toamplify human β2AR from human cDNA to create SEQ ID NO.: 203. Using SalIand PstI a translational fusion was made between β2AR and ToxR (SEQ IDNO.: 202) to create SEQ ID NO.: 204.

[1226] Using oligonucleotides SEQ ID NOS.: 197 and 204 transcriptionalfusions were created between the β2AR-ToxR translational fusion (SEQ IDNO.: 204) and the GS1α:-ToxR translational fusion (SEQ ID NO.: 197) tocreate SEQ ID NO.: 205.

[1227] Oligonucleotides SEQ ID NOS.: 240, 241, 242 and 243 were used toamplify the ctx promoter region (Pctx) from Vibrio cholerae to createSEQ ID NO.: 206. Combining this PCR product in combination with the SEQID NO.: 207 PCR product and amplifying in the presence of SEQ ID NOS.:242, 247, SEQ ID NO.: 208 was created. Using XbaI, the SEQ ID NO.: 208reporter construct was subsequently cloned into pACYC184 forco-transformation with the GPCR-G-protein fusions constructs above.

[1228] Oligonucleotides SEQ ID NOS.: 244, 245, 246 and 247 were used toamplify the lacZ from E. coli to create SEQ ID NO.: 207. Combining thisPCR product in combination with the SEQ ID NO.: 206 PCR product andamplifying in the presence of SEQ ID NOS.: 242 and 247, SEQ ID NO.: 208was created. Using XbaI, the 208 reporter construct was subsequentlycloned into pACYC184 for co-transformation with the GPCR-G-proteinfusions constructs above.

Example 23 Modular Membrane-Targeting and Solubilization ExpressionConstructs

[1229] To produce membrane proteins efficiently in minicells it may benecessary to create chimeric fusions with the membrane protein ofinterest. In this Example various regions of the MalE protein have beencloned into a modular expression system designed to create chimericfusions with direct difficult to target membrane proteins to produceleader domains that will direct the proteins to the cytoplasmic membrane( Miller, K., W., et al. 1998. Production of active chimeric pediocinAcH in Escherichia coli in the absence of processing and secretion genesfrom the Pediococcus pap operon. Appl. Environ. Microbiol. 64:14-20).Similarly, a modified version of the TrxA protein has also been clonedinto this modular expression system to create chimeric fusions withproteins that are difficult to maintain in a soluble conformation(LaVallie, E. R., et al. 1993. A thioredoxin gene fusion expressionsystem that circumvents inclusion body formation in the E. colicytoplasm. Biotechnology (N.Y.) 11:187-193). Table 31 describes each ofthese modular constructs. TABLE 31 MODULAR MEMBRANE-TARGETING ANDSOLUBILIZATION EXPRESSION CONSTRUCTS Protein ^(a) Construct ^(a) SEQ IDNO MalE (1-28) NsiI-MalE(1-28)-Factor Xa-PstI, SalI, XbaI-FLAG, NheI 248MalE (1-370, del 354- NsiI-MalE(1-370, del 354-364)-Factor Xa-PstI,SalI, XbaI-FLAG, 249 364) NheI TrxA (2-109, del 103- PstI, SalI,XbaI-TrxA(2-109, del 103-107)-FLAG-NheI 250 107) MalE (1-28)-TrxA (2-NsiI-MalE(1-28)-Factor Xa-PstI, SalI, XbaI-TrxA (2-109 del 103- 251 109,del 103-107) 107)-FLAG, NheI MalE (1-370, del 354- NsiI-MalE(1-370, del354-364)-Factor Xa-PstI, SalI, XbaI-TrxA (2- 252 364)-TrxA (2-109, del109 del 103-107)-FLAG, NheI 103-107)

[1230] TABLE 32 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE 31. SEQ IDNO.: Primer name 5′to 3′ sequence 253 MalE-1-NsiICGCGGATGCATATGAAAATAAAAACAGGTGCACGCATCCTCGCATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCC 254 MalE-2-middleCGTCGACCGAGGCCTGCAGGCGGGCTTCGATGATTTTGGCGAG AGCCGAGGCGGAAAACATCATCGTCG255 MalE-3s-NheI CGAAGCCCGCCTGCAGGCCTCGGTCGACGCCGAATCTAGAGATTATAAAGATGACGATGACAAATAATAAGCTAGCGGCGC 256 MalE-4-NheIGCGCCGCTAGCTTATTATTTGTCATCG 257 MalE-1a GGTGCACGCATCCTCGCATTATCCGC 258MalE-2a GGCGTTTTCCATGGTGGCGGCAATACGTGG 259 MalE-1-NsiICGCGGATGCATATGAAAATAAAAACAGGTGCACGCATCCTC GCATTATCCGC 260 MalE-2-NheICCGAGGCCTGCAGGCGGGCTTCGATACGCACGGCATACCAGAAAGCGGACTGGGCGTTTTCCATGGTGGCGGCAATACGTGG 261 MalE-3L-NheIGCGCCGCTAGCTTATTATTTGTCATCGTCATCTTTATAATCTCTAGATTCGGCGTCGACCGAGGCCTGCAGGCGGGCTTCGATA CGC 262 TrxA-1aCCTGACTGACGACAGTTTTGACACGG 263 TrxA-2a CCTTTAGACAGTGCACCCACTTTGGTTGCCGC264 TrxA-1a-PstI CGCGGCTGCAGGCCTCGGTCGACGCCGAATCTAGAAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGG 265 TrxA-2-NheIGCGCCGCTAGCTTATTATTTGTCATCGTCATCTTTATAATCCGCCAGGTTCTCTTTCAACTGACCTTTAGACAGTGCACCCACTTT GGTTGCCGC

[1231] Oligonucleotides SEQ ID NOS.: 253, 254, 255 and 256 overlap witheach other to form a scaffold template to PCR amplify malE (1-28) tocreate a SEQ ID NO.: 248. Following PCR amplification, SEQ ID NO.: 248was digested with NsiI and NheI and cloned into SEQ ID NOS.: 152, 154,139 and 141 digested with PstI and XbaI. The resultant products createSEQ ID NOS.: 266, 267, 268 and 269, respectively, that lose both the5-prime PstI and 3-prime XbaI restriction site and retain the PstI,SalI, and XbaI restriction sites between MalE (1-28) and the FLAGsequence. Insertion of a protein in alignment with these sites resultsin a chimeric protein containing amino-terminal MalE (1-28) andcarboxy-terminal FLAG.

[1232] Oligonucleotides SEQ ID NOS.: 257, 258, 259 and 260 were used toamplify malE (1-370 with a deletion removing residues 354-364) to createSEQ ID NO.: 249. Following PCR amplification, SEQ ID NO.: 249 wasdigested with NsiI and NheI and cloned into SEQ ID NOS.: 152, 154, 139and 141 digested with PstI and XbaI. The resultant products create SEQID NOS.: 270, 271, 272 and 273, respectively, that lose both the 5-primePstI and 3-prime XbaI restriction site and retain the PstI, SalI, andXbaI restriction sites between MalE (1-370, del 354-364) and the FLAGsequence. Insertion of a protein in alignment with these sites resultsin a chimeric protein containing amino-terminal MalE (1-370, del354-364) and carboxy-terminal FLAG.

[1233] Oligonucleotides SEQ ID NOS.: 262, 263, 264 and 265 were used toamplify trxA (2-109 with a deletion removing residues 103-107) to createSEQ ID NO.: 250. Following PCR amplification, SEQ ID NO.: 250 wasdigested with PstI and NheI and cloned into SEQ ID NOS.: 152, 154, 139and 141 digested with PstI and XbaI. to create SEQ ID NOS.: 274, 275,276 and 277, respectively. Using these restriction digestioncombinations results in loss of the XbaI SEQ ID NO.: 249 insertion site.

[1234] The resultant products create SEQ ID NOS.: 274, 275, 276 and 277,respectively, that lose the 3-prime XbaI restriction site and retain thePstI, SalI, and XbaI restriction sites on the 3-prime end of the TrxA(1-109, del 103-107) sequence. Insertion of a protein in alignment withthese sites results in a chimeric protein containing Carboxy-terminalTrxA (1-109, del 103-107)-FLAG.

[1235] SEQ ID NO.: 248 was digested with NsiI and XbaI and cloned intoSEQ ID NOS.: 274, 275, 276 and 277 that were digested with PstI andXbaI. The resultant products create SEQ ID NOS.: 278, 279, 280 and 281,respectively, that lose the 5 prime PstI restriction site and retain thePstI, SalI, and XbaI restriction sites between MalE (1-28) and TrxA(1-109, del 103-107). Insertion of a protein in alignment with thesesites results in a chimeric protein containing amino-terminal MalE(1-28) and carboxy-terminal TrxA (1-109, del 103-107)-FLAG.

[1236] SEQ ID NO.: 249 was digested with NsiI and XbaI and cloned intoSEQ ID NOS.: 274, 275, 276 and 277 that were digested with PstI andXbaI. The resultant products create SEQ ID NOS.: 282, 283, 284 and 285,respectively, that lose the 5 prime PstI restriction site and retain thePstI, SalI, and XbaI restriction sites between MalE (1-370, del 354-364)and TrxA (1-109, del 103-107). Insertion of a protein in alignment withthese sites results in a chimeric protein containing amino-terminal MalE(1-370, del 354-364) and carboxy-terminal TrxA (1-109, del103-107)-FLAG.

Example 24 Poroplast™ Formation

[1237] Minicells are used to prepare Poroplasts in order to increase theaccessibility of a membrane protein component and/or domain to theoutside environment. Membrane proteins in the inner membrane areaccessible for ligand binding and/or other interactions in poroplasts,due to the absence of an outer membrane. The removal of the outermembrane from E. coli whole cells and minicells to produce poroplastswas carried out using modifications of previously described protoplastand analysis protocols (Birdsell et al., Production and Ultrastructureof Lysozyme and Ethylenediaminetetraacetate-Lysozyme Spheroplasts ofEscherichia coli, J. Bacteriol. 93:427-437, 1967; Weiss et al.,Protoplast Formation in Escherichia Coli, J. Bacteriol. 128:668-670,1976; Matsuyama, S-I., et al. SecD is involved in the release oftranslocated secretory proteins from the cytoplasmic membrane ofEscherichia coli. 12:265-270, 1993).

[1238] In brief, cells were grown to late-log phase and pelleted at roomtemperature. Minicells were also isolated from cultures in late-logphase. The pellet was washed twice with 50 mM Tris, pH 8.0. Followingthe second wash, 1×10⁹ cells were resuspended in 1 ml 50 mM Tris (pH8.0) that contained 8% sucrose and 2 mM EDTA. Cell/EDTA/sucrose mixtureswere incubated at 37° C. for 10 min, centrifuged, decanted, andporoplasted cells were resuspended in 50 mM Tris, pH 8.0 with 8%sucrose. Incubation with anti-LPS-coated magnetic beads, as described inExample 14, is used to enrich for poroplasts that lack LPS. Followingincubation with the resuspended protoplasted cells, the anti-LPSmagnetic beads were removed from suspension with a magnet.

[1239] To examine the range of molecular sizes that can pass through thecell wall, an IgG molecule was tested for its ability to pass the intactcell wall. Binding of an antibody to the ToxR-PhoA chimera expressed onthe inner membrane minicell poroplasts was measured. Briefly, minicellporoplasts with and without inner membrane-bound ToxR-PhoA wereincubated at 37° C. with anti-PhoA antibody in reaction buffer (50 mMTris, pH 8.0, 8% sucrose, 1% BSA, and 0.01% Tween-20). Followingincubation, poroplasts were centrifuged, washed 3 times with reactionbuffer, and resuspended in 50 mM Tris, pH 8.0 with 8% sucrose. Followingresuspension, bound proteins from 5×10⁷ minicells or minicell poroplastswere separated using denaturing SDS-PAGE, transferred to nitrocellulose,and developed using with both anti-PhoA antibody and secondary antibodyagainst both heavy and light chains of anti-PhoA IgG (Table 33). TABLE33 ANTI-PHOA ACCESSIBILITY TO POROPLAST INNER MEMBRANE-BOUND TOXR-PHOAEDTA (mM) 0 2 0 2 Lysozyme (mg/ml) 0 0 5 5 Poroplasts (ng antibodybound) Protoplasts (ng antibody bound) Minicells ToxR- ND ^(a) 0.6 ND^(a) 12.8 PhoA Minicells only ND ^(a) ND ^(a) ND ^(a) ND ^(a)

[1240] These results demonstrate that the cell wall present onporoplasts is penetrable by an IgG molecule and that an IgG molecule iscapable of passing the intact cell wall and binding to an inner membraneprotein. From this data it appeats that poroplast operate at ˜10% theefficiency of protoplasts by allowing 0.6 ng of IgG to bind innermembrane-bound ToxR-PhoA compared to 12.8 ng. However, given the largesize of IgG (˜150,000 Daltons) it is expected that molecules having asmaller molecular weight will efficiently access inner membrane proteinsin poroplasts.

Example 25 Production of Neurotensin Receptor (NTR)

[1241] To demonstrate expression of NTR in isolated minicells,MalE(L)-NTR (SEQ ID NO.: 166 was cloned into pMPX-67 (SEQ ID NO.: 151).Following minicell isolation, 1.5×10⁹ minicells were induced with 1 mMRhamnose for 2 hour at 37° C. Following induction, the protein producedwas visualized via Western analysis using an anti-MalE antibodyfollowing separation on an SDS-PAGE. The results are shown in FIG. 2.

[1242] These data demonstrates that MalE(L)-NTR is induced 87-fold byaddition of 1 mM rhamnose to the minicell induction mixture.Cross-reactive proteins are host MalE and non-specific binding byGoat-anti-mouse HRP secondary antibody.

[1243] The contents of the articles, patents, and patent applications,and all other documents and electronically available informationmentioned or cited herein, are hereby incorporated by reference in theirentirety to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.Applicants reserve the right to physically incorporate into thisapplication any and all materials and information from any sucharticles, patents, patent applications, or other documents.

[1244] The inventions illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising”, “including,” “containing”, etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the inventions embodied thereinherein disclosed may be resorted to by those skilled in the art, andthat such modifications and variations are considered to be within thescope of this invention.

[1245] The invention has been described broadly and generically herein.Each of the narrower species and subgeneric groupings falling within thegeneric disclosure also form part of the invention. This includes thegeneric description of the invention with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

[1246] Other embodiments are within the following claims. In addition,where features or aspects of the invention are described in terms ofMarkush groups, those skilled in the art will recognize that theinvention is also thereby described in terms of any individual member orsubgroup of members of the Markush group.

1 257 1 1260 DNA E. coli 1 ccatacccgt ttttttgggc tagcaggagg aattcaccctgcagatgttt gaaccaatgg 60 aacttaccaa tgacgcggtg attaaagtca tcggcgtcggcggcggcggc ggtaatgctg 120 ttgaacacat ggtgcgcgag cgcattgaag gtgttgaattcttcgcggta aataccgatg 180 cacaagcgct gcgtaaaaca gcggttggac agacgattcaaatcggtagc ggtatcacca 240 aaggactggg cgctggcgct aatccagaag ttggccgcaatgcggctgat gaggatcgcg 300 atgcattgcg tgcggcgctg gaaggtgcag acatggtctttattgctgcg ggtatgggtg 360 gtggtaccgg tacaggtgca gcaccagtcg tcgctgaagtggcaaaagat ttgggtatcc 420 tgaccgttgc tgtcgtcact aagcctttca actttgaaggcaagaagcgt atggcattcg 480 cggagcaggg gatcactgaa ctgtccaagc atgtggactctctgatcact atcccgaacg 540 acaaactgct gaaagttctg ggccgcggta tctccctgctggatgcgttt ggcgcagcga 600 acgatgtact gaaaggcgct gtgcaaggta tcgctgaactgattactcgt ccgggtttga 660 tgaacgtgga ctttgcagac gtacgcaccg taatgtctgagatgggctac gcaatgatgg 720 gttctggcgt ggcgagcggt gaagaccgtg cggaagaagctgctgaaatg gctatctctt 780 ctccgctgct ggaagatatc gacctgtctg gcgcgcgcggcgtgctggtt aacatcacgg 840 cgggcttcga cctgcgtctg gatgagttcg aaacggtaggtaacaccatc cgtgcatttg 900 cttccgacaa cgcgactgtg gttatcggta cttctcttgacccggatatg aatgacgagc 960 tgcgcgtaac cgttgttgcg acaggtatcg gcatggacaaacgtcctgaa atcactctgg 1020 tgaccaataa gcaggttcag cagccagtga tggatcgctaccagcagcat gggatggctc 1080 cgctgaccca ggagcagaag ccggttgcta aagtcgtgaatgacaatgcg ccgcaaactg 1140 cgaaagagcc ggattatctg gatatcccag cattcctgcgtaagcaagct gattaataat 1200 ctagaggatc cccgggtacc gagctcgaat tcgtaatcatggtcatagct gtttcctgtg 1260 2 1260 DNA E. coli 2 gaattcaggc gctttttagactggtcgtaa tgaaattcag caggatcaca ttctgcagat 60 gtttgaacca atggaacttaccaatgacgc ggtgattaaa gtcatcggcg tcggcggcgg 120 cggcggtaat gctgttgaacacatggtgcg cgagcgcatt gaaggtgttg aattcttcgc 180 ggtaaatacc gatgcacaagcgctgcgtaa aacagcggtt ggacagacga ttcaaatcgg 240 tagcggtatc accaaaggactgggcgctgg cgctaatcca gaagttggcc gcaatgcggc 300 tgatgaggat cgcgatgcattgcgtgcggc gctggaaggt gcagacatgg tctttattgc 360 tgcgggtatg ggtggtggtaccggtacagg tgcagcacca gtcgtcgctg aagtggcaaa 420 agatttgggt atcctgaccgttgctgtcgt cactaagcct ttcaactttg aaggcaagaa 480 gcgtatggca ttcgcggagcaggggatcac tgaactgtcc aagcatgtgg actctctgat 540 cactatcccg aacgacaaactgctgaaagt tctgggccgc ggtatctccc tgctggatgc 600 gtttggcgca gcgaacgatgtactgaaagg cgctgtgcaa ggtatcgctg aactgattac 660 tcgtccgggt ttgatgaacgtggactttgc agacgtacgc accgtaatgt ctgagatggg 720 ctacgcaatg atgggttctggcgtggcgag cggtgaagac cgtgcggaag aagctgctga 780 aatggctatc tcttctccgctgctggaaga tatcgacctg tctggcgcgc gcggcgtgct 840 ggttaacatc acggcgggcttcgacctgcg tctggatgag ttcgaaacgg taggtaacac 900 catccgtgca tttgcttccgacaacgcgac tgtggttatc ggtacttctc ttgacccgga 960 tatgaatgac gagctgcgcgtaaccgttgt tgcgacaggt atcggcatgg acaaacgtcc 1020 tgaaatcact ctggtgaccaataagcaggt tcagcagcca gtgatggatc gctaccagca 1080 gcatgggatg gctccgctgacccaggagca gaagccggtt gctaaagtcg tgaatgacaa 1140 tgcgccgcaa actgcgaaagagccggatta tctggatatc ccagcattcc tgcgtaagca 1200 agctgattaa taatctagaggatccccggg taccgagctc gaattcgtaa tcatggtcat 1260 3 2544 DNA ArtificialSequence Fusion protein 3 aagcctgcat tgcggcgctt cagtctccgc tgcatactgtcccgttacca attatgacaa 60 cttgacggct acatcattca ctttttcttc acaaccggcacggaactcgc tcgggctggc 120 cccggtgcat tttttaaata cccgcgagaa atagagttgatcgtcaaaac caacattgcg 180 accgacggtg gcgataggca tccgggtggt gctcaaaagcagcttcgcct ggctgatacg 240 ttggtcctcg cgccagctta agacgctaat ccctaactgctggcggaaaa gatgtgacag 300 acgcgacggc gacaagcaaa catgctgtgc gacgctggcgatatcaaaat tgctgtctgc 360 caggtgatcg ctgatgtact gacaagcctc gcgtacccgattatccatcg gtggatggag 420 cgactcgtta atcgcttcca tgcgccgcag taacaattgctcaagcagat ttatcgccag 480 cagctccgaa tagcgccctt ccccttgccc ggcgttaatgatttgcccaa acaggtcgct 540 gaaatgcggc tggtgcgctt catccgggcg aaagaaccccgtattggcaa atattgacgg 600 ccagttaagc cattcatgcc agtaggcgcg cggacgaaagtaaacccact ggtgatacca 660 ttcgcgagcc tccggatgac gaccgtagtg atgaatctctcctggcggga acagcaaaat 720 atcacccggt cggcaaacaa attctcgtcc ctgatttttcaccaccccct gaccgcgaat 780 ggtgagattg agaatataac ctttcattcc cagcggtcggtcgataaaaa aatcgagata 840 accgttggcc tcaatcggcg ttaaacccgc caccagatgggcattaaacg agtatcccgg 900 cagcagggga tcattttgcg cttcagccat acttttcatactcccgccat tcagagaaga 960 aaccaattgt ccatattgca tcagacattg ccgtcactgcgtcttttact ggctcttctc 1020 gctaaccaaa ccggtaaccc cgcttattaa aagcattctgtaacaaagcg ggaccaaagc 1080 catgacaaaa acgcgtaaca aaagtgtcta taatcacggcagaaaagtcc acattgatta 1140 tttgcacggc gtcacacttt gctatgccat agcatttttatccataagat tagcggatcc 1200 tacctgacgc tttttatcgc aactctctac tgtttctccatacccgtttt tttgggctag 1260 caggaggaat tcaccctgca gatgtttgaa ccaatggaacttaccaatga cgcggtgatt 1320 aaagtcatcg gcgtcggcgg cggcggcggt aatgctgttgaacacatggt gcgcgagcgc 1380 attgaaggtg ttgaattctt cgcggtaaat accgatgcacaagcgctgcg taaaacagcg 1440 gttggacaga cgattcaaat cggtagcggt atcaccaaaggactgggcgc tggcgctaat 1500 ccagaagttg gccgcaatgc ggctgatgag gatcgcgatgcattgcgtgc ggcgctggaa 1560 ggtgcagaca tggtctttat tgctgcgggt atgggtggtggtaccggtac aggtgcagca 1620 ccagtcgtcg ctgaagtggc aaaagatttg ggtatcctgaccgttgctgt cgtcactaag 1680 cctttcaact ttgaaggcaa gaagcgtatg gcattcgcggagcaggggat cactgaactg 1740 tccaagcatg tggactctct gatcactatc ccgaacgacaaactgctgaa agttctgggc 1800 cgcggtatct ccctgctgga tgcgtttggc gcagcgaacgatgtactgaa aggcgctgtg 1860 caaggtatcg ctgaactgat tactcgtccg ggtttgatgaacgtggactt tgcagacgta 1920 cgcaccgtaa tgtctgagat gggctacgca atgatgggttctggcgtggc gagcggtgaa 1980 gaccgtgcgg aagaagctgc tgaaatggct atctcttctccgctgctgga agatatcgac 2040 ctgtctggcg cgcgcggcgt gctggttaac atcacggcgggcttcgacct gcgtctggat 2100 gagttcgaaa cggtaggtaa caccatccgt gcatttgcttccgacaacgc gactgtggtt 2160 atcggtactt ctcttgaccc ggatatgaat gacgagctgcgcgtaaccgt tgttgcgaca 2220 ggtatcggca tggacaaacg tcctgaaatc actctggtgaccaataagca ggttcagcag 2280 ccagtgatgg atcgctacca gcagcatggg atggctccgctgacccagga gcagaagccg 2340 gttgctaaag tcgtgaatga caatgcgccg caaactgcgaaagagccgga ttatctggat 2400 atcccagcat tcctgcgtaa gcaagctgat taataatctagaggcgttac caattatgac 2460 aacttgacgg gaagttccta tactttctag agaataggaacttcccaaag ccagtatcaa 2520 ctcagacaaa ggcaaagcat cttg 2544 4 3350 DNAArtificial Sequence Fusion protein 4 aagcctgcat tgcggcgctt cagtctccgctgcatactgt ccttaatctt tctgcgaatt 60 gagatgacgc cactggctgg gcgtcatcccggtttcccgg gtaaacacca ccgaaaaata 120 gttactatct tcaaagccac attcggtcgaaatatcactg attaacaggc ggctatgctg 180 gagaagatat tgcgcatgac acactctgacctgtcgcaga tattgattga tggtcattcc 240 agtctgctgg cgaaattgct gacgcaaaacgcgctcactg cacgatgcct catcacaaaa 300 tttatccagc gcaaagggac ttttcaggctagccgccagc cgggtaatca gcttatccag 360 caacgtttcg ctggatgttg gcggcaacgaatcactggtg taacgatggc gattcagcaa 420 catcaccaac tgcccgaaca gcaactcagccatttcgtta gcaaacggca catgctgact 480 actttcatgc tcaagctgac cgataacctgccgcgcctgc gccatcccca tgctacctaa 540 gcgccagtgt ggttgccctg cgctggcgttaaatcccgga atcgccccct gccagtcaag 600 attcagcttc agacgctccg ggcaataaataatattctgc aaaaccagat cgttaacgga 660 agcgtaggag tgtttatcgt cagcatgaatgtaaaagaga tcgccacggg taatgcgata 720 agggcgatcg ttgagtacat gcaggccattaccgcgccag acaatcacca gctcacaaaa 780 atcatgtgta tgttcagcaa agacatcttgcggataacgg tcagccacag cgactgcctg 840 ctggtcgctg gcaaaaaaat catctttgagaagttttaac tgatgcgcca ccgtggctac 900 ctcggccaga gaacgaagtt gattattcgcaatatggcgt acaaatacgt tgagaagatt 960 cgcgttattg cagaaagcca tcccgtccctggcgaatatc acgcggtgac cagttaaact 1020 ctcggcgaaa aagcgtcgaa aagtggttactgtcgctgaa tccacagcga taggcgatgt 1080 cagtaacgct ggcctcgctg tggcgtagcagatgtcgggc tttcatcagt cgcaggcggt 1140 tcaggtatcg ctgaggcgtc agtcccgtttgctgcttaag ctgccgatgt agcgtacgca 1200 gtgaaagaga aaattgatcc gccacggcatcccaattcac ctcatcggca aaatggtcct 1260 ccagccaggc cagaagcaag ttgagacgtgatgcgctgtt ttccaggttc tcctgcaaac 1320 tgcttttacg cagcaagagc agtaattgcataaacaagat ctcgcgactg gcggtcgagg 1380 gtaaatcatt ttccccttcc tgctgttccatctgtgcaac cagctgtcgc acctgctgca 1440 atacgctgtg gttaacgcgc cagtgagacggatactgccc atccagctct tgtggcagca 1500 actgattcag cccggcgaga aactgaaatcgatccggcga gcgatacagc acattggtca 1560 gacacagatt atcggtatgt tcatacagatgccgatcatg atcgcgtacg aaacagaccg 1620 tgccaccggt gatggtatag ggctgcccattaaacacatg aatacccgtg ccatgttcga 1680 caatcacaat ttcatgaaaa tcatgatgatgttcaggaaa atccgcctgc gggagccggg 1740 gttctatcgc cacggacgcg ttaccagacggaaaaaaatc cacactatgt aatacggtca 1800 tactggcctc ctgatgtcgt caacacggcgaaatagtaat cacgaggtca ggttcttacc 1860 ttaaattttc gacggaaaac cacgtaaaaaacgtcgattt ttcaagatac agcgtgaatt 1920 ttcaggaaat gcggtgagca tcacatcaccacaattcagc aaattgtgaa catcatcacg 1980 ttcatctttc cctggttgcc aatggcccattttcctgtca gtaacgagaa ggtcgcgaat 2040 tcaggcgctt tttagactgg tcgtaatgaaattcagcagg atcacatatg tttgaaccaa 2100 tggaacttac caatgacgcg gtgattaaagtcatcggcgt cggcggcggc ggcggtaatg 2160 ctgttgaaca catggtgcgc gagcgcattgaaggtgttga attcttcgcg gtaaataccg 2220 atgcacaagc gctgcgtaaa acagcggttggacagacgat tcaaatcggt agcggtatca 2280 ccaaaggact gggcgctggc gctaatccagaagttggccg caatgcggct gatgaggatc 2340 gcgatgcatt gcgtgcggcg ctggaaggtgcagacatggt ctttattgct gcgggtatgg 2400 gtggtggtac cggtacaggt gcagcaccagtcgtcgctga agtggcaaaa gatttgggta 2460 tcctgaccgt tgctgtcgtc actaagcctttcaactttga aggcaagaag cgtatggcat 2520 tcgcggagca ggggatcact gaactgtccaagcatgtgga ctctctgatc actatcccga 2580 acgacaaact gctgaaagtt ctgggccgcggtatctccct gctggatgcg tttggcgcag 2640 cgaacgatgt actgaaaggc gctgtgcaaggtatcgctga actgattact cgtccgggtt 2700 tgatgaacgt ggactttgca gacgtacgcaccgtaatgtc tgagatgggc tacgcaatga 2760 tgggttctgg cgtggcgagc ggtgaagaccgtgcggaaga agctgctgaa atggctatct 2820 cttctccgct gctggaagat atcgacctgtctggcgcgcg cggcgtgctg gttaacatca 2880 cggcgggctt cgacctgcgt ctggatgagttcgaaacggt aggtaacacc atccgtgcat 2940 ttgcttccga caacgcgact gtggttatcggtacttctct tgacccggat atgaatgacg 3000 agctgcgcgt aaccgttgtt gcgacaggtatcggcatgga caaacgtcct gaaatcactc 3060 tggtgaccaa taagcaggtt cagcagccagtgatggatcg ctaccagcag catgggatgg 3120 ctccgctgac ccaggagcag aagccggttgctaaagtcgt gaatgacaat gcgccgcaaa 3180 ctgcgaaaga gccggattat ctggatatcccagcattcct gcgtaagcaa gctgattaat 3240 aatctagagg cgttaccaat tatgacaacttgacgggaag ttcctatact ttctagagaa 3300 taggaacttc ccaaagccag tatcaactcagacaaaggca aagcatcttg 3350 5 2280 DNA Artificial Sequence Fusion protein5 aagcctgcat tgcggcgctt cagtctccgc tgcatactgt ccttaataaa gtgagtcgat 60attgtctttg ttgaccagta ataccttatg gaaacggata attcgcttat ccatatctac 120gtcggcctta cccagattct gcatttctaa tccaggcttg atctcttcac ccttcagcaa 180cgtgctggcg acggctgcga gtgcgtaacc tgcagaggcc ggatcgtaag taatcccttc 240ggtgatatca ccacttttaa tcagtgatgc cgcctgtgaa gggatcatca tgccatagac 300tgcgacttta tttttcgccc gtttctcttt caccgcacgt cccgcgccaa tcggaccgtt 360tgaaccaaag gagacaaccg ctttcaagtc aggataggtt ttcatcaggt ccagtgtagt 420acgacgtgag acatccacac tctcggcaac cggcatgcgg cgggtaactt catgcatatc 480cgggtaatgc tctttctggt atttcaccag caagtcagcc cataagttat gctgcggcac 540ggtcaaacta cccacgtaaa tcacatagcc gcccttgcca cccatgcgtt tcgccatatg 600ctcaacatat tcagcggcaa atttttcgtt atcaatgatt tcgatatccc agttagcact 660tggctgaccg ggggattcgt tggtcagaac cacaattccg gcatctcgcg cttttttgaa 720taccggttcc agcacgttgg catcgtttgg cacgatagta attgcattaa ccttacgggc 780gattaaatcc tcaataattt taacttgttg cggagcatca gtacttgaag gccccacctg 840tgaggcatta acaccaaagg ctttacccgc ctcaaccaca ccttcgccca tgcgattaaa 900ccacggcata ccatcgactt tagaaatatt caccacgact ttttccgctg cctggagcgg 960cgcagaaatt agcgcagcgc ctaataacag cgaagacacc atattgataa caaaacgttt 1020attcatcata tggaacttac caatgacgcg gtgattaaag tcatcggcgt cggcggcggc 1080ggcggtaatg ctgttgaaca catggtgcgc gagcgcattg aaggtgttga attcttcgcg 1140gtaaataccg atgcacaagc gctgcgtaaa acagcggttg gacagacgat tcaaatcggt 1200agcggtatca ccaaaggact gggcgctggc gctaatccag aagttggccg caatgcggct 1260gatgaggatc gcgatgcatt gcgtgcggcg ctggaaggtg cagacatggt ctttattgct 1320gcgggtatgg gtggtggtac cggtacaggt gcagcaccag tcgtcgctga agtggcaaaa 1380gatttgggta tcctgaccgt tgctgtcgtc actaagcctt tcaactttga aggcaagaag 1440cgtatggcat tcgcggagca ggggatcact gaactgtcca agcatgtgga ctctctgatc 1500actatcccga acgacaaact gctgaaagtt ctgggccgcg gtatctccct gctggatgcg 1560tttggcgcag cgaacgatgt actgaaaggc gctgtgcaag gtatcgctga actgattact 1620cgtccgggtt tgatgaacgt ggactttgca gacgtacgca ccgtaatgtc tgagatgggc 1680tacgcaatga tgggttctgg cgtggcgagc ggtgaagacc gtgcggaaga agctgctgaa 1740atggctatct cttctccgct gctggaagat atcgacctgt ctggcgcgcg cggcgtgctg 1800gttaacatca cggcgggctt cgacctgcgt ctggatgagt tcgaaacggt aggtaacacc 1860atccgtgcat ttgcttccga caacgcgact gtggttatcg gtacttctct tgacccggat 1920atgaatgacg agctgcgcgt aaccgttgtt gcgacaggta tcggcatgga caaacgtcct 1980gaaatcactc tggtgaccaa taagcaggtt cagcagccag tgatggatcg ctaccagcag 2040catgggatgg ctccgctgac ccaggagcag aagccggttg ctaaagtcgt gaatgacaat 2100gcgccgcaaa ctgcgaaaga gccggattat ctggatatcc cagcattcct gcgtaagcaa 2160gctgattaat aatctagagg cgttaccaat tatgacaact tgacgggaag ttcctattct 2220ctagaaagta taggaacttc ccaaagccag tatcaactca gacaaaggca aagcatcttg 2280 64728 DNA E. coli 6 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacatgcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccgtcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcagagcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggagaaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatcggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgattaagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaagcttaattaa tctttctgcg 420 aattgagatg acgccactgg ctgggcgtca tcccggtttcccgggtaaac accaccgaaa 480 aatagttact atcttcaaag ccacattcgg tcgaaatatcactgattaac aggcggctat 540 gctggagaag atattgcgca tgacacactc tgacctgtcgcagatattga ttgatggtca 600 ttccagtctg ctggcgaaat tgctgacgca aaacgcgctcactgcacgat gcctcatcac 660 aaaatttatc cagcgcaaag ggacttttca ggctagccgccagccgggta atcagcttat 720 ccagcaacgt ttcgctggat gttggcggca acgaatcactggtgtaacga tggcgattca 780 gcaacatcac caactgcccg aacagcaact cagccatttcgttagcaaac ggcacatgct 840 gactactttc atgctcaagc tgaccgataa cctgccgcgcctgcgccatc cccatgctac 900 ctaagcgcca gtgtggttgc cctgcgctgg cgttaaatcccggaatcgcc ccctgccagt 960 caagattcag cttcagacgc tccgggcaat aaataatattctgcaaaacc agatcgttaa 1020 cggaagcgta ggagtgttta tcgtcagcat gaatgtaaaagagatcgcca cgggtaatgc 1080 gataagggcg atcgttgagt acatgcaggc cattaccgcgccagacaatc accagctcac 1140 aaaaatcatg tgtatgttca gcaaagacat cttgcggataacggtcagcc acagcgactg 1200 cctgctggtc gctggcaaaa aaatcatctt tgagaagttttaactgatgc gccaccgtgg 1260 ctacctcggc cagagaacga agttgattat tcgcaatatggcgtacaaat acgttgagaa 1320 gattcgcgtt attgcagaaa gccatcccgt ccctggcgaatatcacgcgg tgaccagtta 1380 aactctcggc gaaaaagcgt cgaaaagtgg ttactgtcgctgaatccaca gcgataggcg 1440 atgtcagtaa cgctggcctc gctgtggcgt agcagatgtcgggctttcat cagtcgcagg 1500 cggttcaggt atcgctgagg cgtcagtccc gtttgctgcttaagctgccg atgtagcgta 1560 cgcagtgaaa gagaaaattg atccgccacg gcatcccaattcacctcatc ggcaaaatgg 1620 tcctccagcc aggccagaag caagttgaga cgtgatgcgctgttttccag gttctcctgc 1680 aaactgcttt tacgcagcaa gagcagtaat tgcataaacaagatctcgcg actggcggtc 1740 gagggtaaat cattttcccc ttcctgctgt tccatctgtgcaaccagctg tcgcacctgc 1800 tgcaatacgc tgtggttaac gcgccagtga gacggatactgcccatccag ctcttgtggc 1860 agcaactgat tcagcccggc gagaaactga aatcgatccggcgagcgata cagcacattg 1920 gtcagacaca gattatcggt atgttcatac agatgccgatcatgatcgcg tacgaaacag 1980 accgtgccac cggtgatggt atagggctgc ccattaaacacatgaatacc cgtgccatgt 2040 tcgacaatca caatttcatg aaaatcatga tgatgttcaggaaaatccgc ctgcgggagc 2100 cggggttcta tcgccacgga cgcgttacca gacggaaaaaaatccacact atgtaatacg 2160 gtcatactgg cctcctgatg tcgtcaacac ggcgaaatagtaatcacgag gtcaggttct 2220 taccttaaat tttcgacgga aaaccacgta aaaaacgtcgatttttcaag atacagcgtg 2280 aattttcagg aaatgcggtg agcatcacat caccacaattcagcaaattg tgaacatcat 2340 cacgttcatc tttccctggt tgccaatggc ccattttcctgtcagtaacg agaaggtcgc 2400 gaattcaggc gctttttaga ctggtcgtaa tgaaattcagcaggatcaca ttctgcaggt 2460 cgactctaga ggatccccgg gtaccgagct cgaattcgtaatcatggtca tagctgtttc 2520 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacatacgagccgga agcataaagt 2580 gtaaagcctg gggtgcctaa tgagtgagct aactcacattaattgcgttg cgctcactgc 2640 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcattaatgaatcggc caacgcgcgg 2700 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctcgctcactgac tcgctgcgct 2760 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaaggcggtaata cggttatcca 2820 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaaaggccagcaa aaggccagga 2880 accgtaaaaa ggccgcgttg ctggcgtttt tccataggctccgcccccct gacgagcatc 2940 acaaaaatcg acgctcaagt cagaggtggc gaaacccgacaggactataa agataccagg 3000 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttccgaccctgccg cttaccggat 3060 acctgtccgc ctttctccct tcgggaagcg tggcgctttctcatagctca cgctgtaggt 3120 atctcagttc ggtgtaggtc gttcgctcca agctgggctgtgtgcacgaa ccccccgttc 3180 agcccgaccg ctgcgcctta tccggtaact atcgtcttgagtccaacccg gtaagacacg 3240 acttatcgcc actggcagca gccactggta acaggattagcagagcgagg tatgtaggcg 3300 gtgctacaga gttcttgaag tggtggccta actacggctacactagaagg acagtatttg 3360 gtatctgcgc tctgctgaag ccagttacct tcggaaaaagagttggtagc tcttgatccg 3420 gcaaacaaac caccgctggt agcggtggtt tttttgtttgcaagcagcag attacgcgca 3480 gaaaaaaagg atctcaagaa gatcctttga tcttttctacggggtctgac gctcagtgga 3540 acgaaaactc acgttaaggg attttggtca tgagattatcaaaaaggatc ttcacctaga 3600 tccttttaaa ttaaaaatga agttttaaat caatctaaagtatatatgag taaacttggt 3660 ctgacagtta ccaatgctta atcagtgagg cacctatctcagcgatctgt ctatttcgtt 3720 catccatagt tgcctgactc cccgtcgtgt agataactacgatacgggag ggcttaccat 3780 ctggccccag tgctgcaatg ataccgcgag acccacgctcaccggctcca gatttatcag 3840 caataaacca gccagccgga agggccgagc gcagaagtggtcctgcaact ttatccgcct 3900 ccatccagtc tattaattgt tgccgggaag ctagagtaagtagttcgcca gttaatagtt 3960 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtcacgctcgtcg tttggtatgg 4020 cttcattcag ctccggttcc caacgatcaa ggcgagttacatgatccccc atgttgtgca 4080 aaaaagcggt tagctccttc ggtcctccga tcgttgtcagaagtaagttg gccgcagtgt 4140 tatcactcat ggttatggca gcactgcata attctcttactgtcatgcca tccgtaagat 4200 gcttttctgt gactggtgag tactcaacca agtcattctgagaatagtgt atgcggcgac 4260 cgagttgctc ttgcccggcg tcaatacggg ataataccgcgccacatagc agaactttaa 4320 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaactctcaaggatc ttaccgctgt 4380 tgagatccag ttcgatgtaa cccactcgtg cacccaactgatcttcagca tcttttactt 4440 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaatgccgcaaaa aagggaataa 4500 gggcgacacg gaaatgttga atactcatac tcttcctttttcaatattat tgaagcattt 4560 atcagggtta ttgtctcatg agcggataca tatttgaatgtatttagaaa aataaacaaa 4620 taggggttcc gcgcacattt ccccgaaaag tgccacctgacgtctaagaa accattatta 4680 tcatgacatt aacctataaa aataggcgta tcacgaggccctttcgtc 4728 7 1440 DNA E. coli Cloning primer 7 gaattcaggc gctttttagactggtcgtaa tgaaattcag caggatcaca ttctgcagat 60 gcctgttctg gaaaaccgggctgctcaggg cgatattact gcacccggcg gtgctcgccg 120 tttaacgggt gatcagactgccgctctgcg tgattctctt agcgataaac ctgcaaaaaa 180 tattattttg ctgattggcgatgggatggg ggactcggaa attactgccg cacgtaatta 240 tgccgaaggt gcgggcggcttttttaaagg tatagatgcc ttaccgctta ccgggcaata 300 cactcactat gcgctgaataaaaaaaccgg caaaccggac tacgtcaccg actcggctgc 360 atcagcaacc gcctggtcaaccggtgtcaa aacctataac ggcgcgctgg gcgtcgatat 420 tcacgaaaaa gatcacccaacgattctgga aatggcaaaa gccgcaggtc tggcgaccgg 480 taacgtttct accgcagagttgcaggatgc cacgcccgct gcgctggtgg cacatgtgac 540 ctcgcgcaaa tgctacggtccgagcgcgac cagtgaaaaa tgtccgggta acgctctgga 600 aaaaggcgga aaaggatcgattaccgaaca gctgcttaac gctcgtgccg acgttacgct 660 tggcggcggc gcaaaaacctttgctgaaac ggcaaccgct ggtgaatggc agggaaaaac 720 gctgcgtgaa caggcacaggcgcgtggtta tcagttggtg agcgatgctg cctcactgaa 780 ttcggtgacg gaagcgaatcagcaaaaacc cctgcttggc ctgtttgctg acggcaatat 840 gccagtgcgc tggctaggaccgaaagcaac gtaccatggc aatatcgata agcccgcagt 900 cacctgtacg ccaaatccgcaacgtaatga cagtgtacca accctggcgc agatgaccga 960 caaagccatt gaattgttgagtaaaaatga gaaaggcttt ttcctgcaag ttgaaggtgc 1020 gtcaatcgat aaacaggatcatgctgcgaa tccttgtggg caaattggcg agacggtcga 1080 tctcgatgaa gccgtacaacgggcgctgga attcgctaaa aaggagggta acacgctggt 1140 catagtcacc gctgatcacgcccacgccag ccagattgtt gcgccggata ccaaagctcc 1200 gggcctcacc caggcgctaaataccaaaga tggcgcagtg atggtgatga gttacgggaa 1260 ctccgaagag gattcacaagaacataccgg cagtcagttg cgtattgcgg cgtatggccc 1320 gcatgccgcc aatgttgttggactgaccga ccagaccgat ctcttctaca ccatgaaagc 1380 cgctctgggg ctgaaataataatctagagg atccccgggt accgagctcg aattcgtaat 1440 8 1560 DNA E. coliCloning primer 8 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caggatcacattctgcagat 60 gtcacggccg agacttatag tcgctttgtt tttatttttt aatgtatttgtacatggaga 120 aaataaagtg aaacaaagca ctattgcact ggcactctta ccgttactgtttacccctgt 180 gacaaaagcc cggacaccag aaatgcctgt tctggaaaac cgggctgctcagggcgatat 240 tactgcaccc ggcggtgctc gccgtttaac gggtgatcag actgccgctctgcgtgattc 300 tcttagcgat aaacctgcaa aaaatattat tttgctgatt ggcgatgggatgggggactc 360 ggaaattact gccgcacgta attatgccga aggtgcgggc ggcttttttaaaggtataga 420 tgccttaccg cttaccgggc aatacactca ctatgcgctg aataaaaaaaccggcaaacc 480 ggactacgtc accgactcgg ctgcatcagc aaccgcctgg tcaaccggtgtcaaaaccta 540 taacggcgcg ctgggcgtcg atattcacga aaaagatcac ccaacgattctggaaatggc 600 aaaagccgca ggtctggcga ccggtaacgt ttctaccgca gagttgcaggatgccacgcc 660 cgctgcgctg gtggcacatg tgacctcgcg caaatgctac ggtccgagcgcgaccagtga 720 aaaatgtccg ggtaacgctc tggaaaaagg cggaaaagga tcgattaccgaacagctgct 780 taacgctcgt gccgacgtta cgcttggcgg cggcgcaaaa acctttgctgaaacggcaac 840 cgctggtgaa tggcagggaa aaacgctgcg tgaacaggca caggcgcgtggttatcagtt 900 ggtgagcgat gctgcctcac tgaattcggt gacggaagcg aatcagcaaaaacccctgct 960 tggcctgttt gctgacggca atatgccagt gcgctggcta ggaccgaaagcaacgtacca 1020 tggcaatatc gataagcccg cagtcacctg tacgccaaat ccgcaacgtaatgacagtgt 1080 accaaccctg gcgcagatga ccgacaaagc cattgaattg ttgagtaaaaatgagaaagg 1140 ctttttcctg caagttgaag gtgcgtcaat cgataaacag gatcatgctgcgaatccttg 1200 tgggcaaatt ggcgagacgg tcgatctcga tgaagccgta caacgggcgctggaattcgc 1260 taaaaaggag ggtaacacgc tggtcatagt caccgctgat cacgcccacgccagccagat 1320 tgttgcgccg gataccaaag ctccgggcct cacccaggcg ctaaataccaaagatggcgc 1380 agtgatggtg atgagttacg ggaactccga agaggattca caagaacataccggcagtca 1440 gttgcgtatt gcggcgtatg gcccgcatgc cgccaatgtt gttggactgaccgaccagac 1500 cgatctcttc tacaccatga aagccgctct ggggctgaaa taatctagaggatccccggg 1560 9 1500 DNA E. coli Cloning primer 9 gaattcaggcgctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcagat 60 gaacttggggaatcgactgt ttattctgat agcggtctta cttcccctcg cagtattact 120 gctcatgcctgttctggaaa accgggctgc tcagggcgat attactgcac ccggcggtgc 180 tcgccgtttaacgggtgatc agactgccgc tctgcgtgat tctcttagcg ataaacctgc 240 aaaaaatattattttgctga ttggcgatgg gatgggggac tcggaaatta ctgccgcacg 300 taattatgccgaaggtgcgg gcggcttttt taaaggtata gatgccttac cgcttaccgg 360 gcaatacactcactatgcgc tgaataaaaa aaccggcaaa ccggactacg tcaccgactc 420 ggctgcatcagcaaccgcct ggtcaaccgg tgtcaaaacc tataacggcg cgctgggcgt 480 cgatattcacgaaaaagatc acccaacgat tctggaaatg gcaaaagccg caggtctggc 540 gaccggtaacgtttctaccg cagagttgca ggatgccacg cccgctgcgc tggtggcaca 600 tgtgacctcgcgcaaatgct acggtccgag cgcgaccagt gaaaaatgtc cgggtaacgc 660 tctggaaaaaggcggaaaag gatcgattac cgaacagctg cttaacgctc gtgccgacgt 720 tacgcttggcggcggcgcaa aaacctttgc tgaaacggca accgctggtg aatggcaggg 780 aaaaacgctgcgtgaacagg cacaggcgcg tggttatcag ttggtgagcg atgctgcctc 840 actgaattcggtgacggaag cgaatcagca aaaacccctg cttggcctgt ttgctgacgg 900 caatatgccagtgcgctggc taggaccgaa agcaacgtac catggcaata tcgataagcc 960 cgcagtcacctgtacgccaa atccgcaacg taatgacagt gtaccaaccc tggcgcagat 1020 gaccgacaaagccattgaat tgttgagtaa aaatgagaaa ggctttttcc tgcaagttga 1080 aggtgcgtcaatcgataaac aggatcatgc tgcgaatcct tgtgggcaaa ttggcgagac 1140 ggtcgatctcgatgaagccg tacaacgggc gctggaattc gctaaaaagg agggtaacac 1200 gctggtcatagtcaccgctg atcacgccca cgccagccag attgttgcgc cggataccaa 1260 agctccgggcctcacccagg cgctaaatac caaagatggc gcagtgatgg tgatgagtta 1320 cgggaactccgaagaggatt cacaagaaca taccggcagt cagttgcgta ttgcggcgta 1380 tggcccgcatgccgccaatg ttgttggact gaccgaccag accgatctct tctacaccat 1440 gaaagccgctctggggctga aataataatc tagaggatcc ccgggtaccg agctcgaatt 1500 10 3908 DNAArtificial Sequence Expression vector 10 tcgcgcgttt cggtgatgacggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggatgccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctggcttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaataccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgcaactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaagggggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tgctcgcctg ctgcgagctgggtaagcgga caaattctca ccgtctccgg tggtggggta 420 caggagctca attaatacactaacggaccg gtaaacaacc gtgcgtgttg tttaccggga 480 taaactcatc aacgtctctgctaaataact ggcagccaaa tcacggctat tggttaacca 540 atttcagagt gaaaagtatacgaatagagt gtgccttcgc actattcaac agcaatgata 600 ggcgctcacc tgacaacgcggtaaactagt tattcacgct aactataatg gtttaatgat 660 ggataacatg cagactgaagcacaaccgac acggacccgg atcctcaatg ctgccagaga 720 gattttttca gaaaatggatttcacagtgc ctcgatgaaa gccatctgta aatcttgcgc 780 cattagtccc gggacgctctatcaccattt catctccaaa gaagccttga ttcaggcgat 840 tatcttacag gaccaggagagggcgctggc ccgtttccgg gaaccgattg aagggattca 900 tttcgttgac tatatggtcgagtccattgt ctctctcacc catgaagcct ttggacaacg 960 ggcgctggtg gttgaaattatggcggaagg gatgcgtaac ccacaggtcg ccgccatgct 1020 taaaaataag catatgacgatcacggaatt tgttgcccag cggatgcgtg atgcccagca 1080 aaaaggcgag ataagcccagacatcaacac ggcaatgact tcacgtttac tgctggatct 1140 gacctacggt gtactggccgatatcgaagc ggaagacctg gcgcgtgaag cgtcgtttgc 1200 tcagggatta cgcgcgatgattggcggtat cttaaccgca tcctgattct ctctcttttt 1260 cggcgggctg gtgataactgtgcccgcgtt tcatatcgta atttctctgt gcaaaaatta 1320 tccttcccgg cttcggagaattccccccaa aatattcact gtagccatat gtcatgagag 1380 tttatcgttc ccaatacgctcgaacgaacg ttcggttgct tattttatgg cttctgtcaa 1440 cgctgtttta aagattaatgcgatctatat cacgctgtgg gtattgcagt ttttggtttt 1500 ttgatcgcgg tgtcagttctttttatttcc atttctcttc catgggtttc tcacagataa 1560 ctgtgtgcaa cacagaattggttaactaat cagattaaag gttgaccagt attattatct 1620 taatgaggag tcctgcaggtcgactctaga ggatccccgg gtaccgagct cgaattcgta 1680 atcatggtca tagctgtttcctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 1740 acgagccgga agcataaagtgtaaagcctg gggtgcctaa tgagtgagct aactcacatt 1800 aattgcgttg cgctcactgcccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 1860 atgaatcggc caacgcgcggggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 1920 gctcactgac tcgctgcgctcggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 1980 ggcggtaata cggttatccacagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 2040 aggccagcaa aaggccaggaaccgtaaaaa ggccgcgttg ctggcgtttt tccataggct 2100 ccgcccccct gacgagcatcacaaaaatcg acgctcaagt cagaggtggc gaaacccgac 2160 aggactataa agataccaggcgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 2220 gaccctgccg cttaccggatacctgtccgc ctttctccct tcgggaagcg tggcgctttc 2280 tcatagctca cgctgtaggtatctcagttc ggtgtaggtc gttcgctcca agctgggctg 2340 tgtgcacgaa ccccccgttcagcccgaccg ctgcgcctta tccggtaact atcgtcttga 2400 gtccaacccg gtaagacacgacttatcgcc actggcagca gccactggta acaggattag 2460 cagagcgagg tatgtaggcggtgctacaga gttcttgaag tggtggccta actacggcta 2520 cactagaagg acagtatttggtatctgcgc tctgctgaag ccagttacct tcggaaaaag 2580 agttggtagc tcttgatccggcaaacaaac caccgctggt agcggtggtt tttttgtttg 2640 caagcagcag attacgcgcagaaaaaaagg atctcaagaa gatcctttga tcttttctac 2700 ggggtctgac gctcagtggaacgaaaactc acgttaaggg attttggtca tgagattatc 2760 aaaaaggatc ttcacctagatccttttaaa ttaaaaatga agttttaaat caatctaaag 2820 tatatatgag taaacttggtctgacagtta ccaatgctta atcagtgagg cacctatctc 2880 agcgatctgt ctatttcgttcatccatagt tgcctgactc cccgtcgtgt agataactac 2940 gatacgggag ggcttaccatctggccccag tgctgcaatg ataccgcgag acccacgctc 3000 accggctcca gatttatcagcaataaacca gccagccgga agggccgagc gcagaagtgg 3060 tcctgcaact ttatccgcctccatccagtc tattaattgt tgccgggaag ctagagtaag 3120 tagttcgcca gttaatagtttgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 3180 acgctcgtcg tttggtatggcttcattcag ctccggttcc caacgatcaa ggcgagttac 3240 atgatccccc atgttgtgcaaaaaagcggt tagctccttc ggtcctccga tcgttgtcag 3300 aagtaagttg gccgcagtgttatcactcat ggttatggca gcactgcata attctcttac 3360 tgtcatgcca tccgtaagatgcttttctgt gactggtgag tactcaacca agtcattctg 3420 agaatagtgt atgcggcgaccgagttgctc ttgcccggcg tcaatacggg ataataccgc 3480 gccacatagc agaactttaaaagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 3540 ctcaaggatc ttaccgctgttgagatccag ttcgatgtaa cccactcgtg cacccaactg 3600 atcttcagca tcttttactttcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 3660 tgccgcaaaa aagggaataagggcgacacg gaaatgttga atactcatac tcttcctttt 3720 tcaatattat tgaagcatttatcagggtta ttgtctcatg agcggataca tatttgaatg 3780 tatttagaaa aataaacaaataggggttcc gcgcacattt ccccgaaaag tgccacctga 3840 cgtctaagaa accattattatcatgacatt aacctataaa aataggcgta tcacgaggcc 3900 ctttcgtc 3908 11 3872DNA E. coli 11 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccggagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcgtcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgtactgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgcatcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcctcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggtaacgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttttagccgggaaacgtc 420 tggcggcgct gttggctaag tttgcggtat tgttgcggcg acatgccgacatatttgccg 480 aacgtgctgt aaaaacgact acttgaacga aagcctgccg tcagggcaatatcgagaata 540 cttttatcgg tatcgctcag taacgcgcga acgtggttga tgcgcatcgcggtaatgtac 600 tgtttcatcg tcaattgcat gacccgctgg aatatcccca ttgcatagttggcgttaagt 660 ttgacgtgct cagccacatc gttgatggtc agcgcctgat catagttttcggcaataaag 720 cccagcatct ggctaacata aaattgcgca tggcgcgaga cgctgtttttgtgtgtgcgc 780 gaggttttat tgaccagaat cggttcccag ccagagaggc taaatcgcttgagcatcagg 840 ccaatttcat caatggcgag ctggcgaatt tgctcgttcg gactgtttaattcctgctgc 900 cagcggcgca cttcaaacgg gctaagttgc tgtgtggcca gtgatttgatcaccatgccg 960 tgagtgacgt ggttaatcag gtctttatcc agcggccagg agagaaacagatgcatcggc 1020 agattaaaaa tcgccatgct ctgacaggtt ccggtatctg ttagttggtgcggtgtacag 1080 gcccagaaca gcgtgatatg accctgattg atattcactt tttcattgttgatcaggtat 1140 tccacatcgc catcgaaagg cacattcact tcgacctgac catgccagtggctggtgggc 1200 atgatatgcg gtgcgcgaaa ctcaatctcc atccgctggt attccgaatacagcgacagc 1260 gggctgcggg tctgtttttc gtcgctgctg cacataaacg tatctgtattcatggatggc 1320 tctctttcct ggaatatcag aattatggca ggagtgaggg aggatgactgcgagtgggag 1380 cacggttttc accctcttcc cagaggggcg aggggactct ccgagtatcatgaggccgaa 1440 aactctgctt ttcaggtaat ttattcccat aaactcagat ttactgctgcttcacgcagg 1500 atctgagttt atgggaatgc tcaacctgga agccggaggt tttctgcagattcgcctgcc 1560 atgatgaagt tattcaagca agccaggaga tctggtaccc gggtcgactctagaggatcc 1620 ccgggtaccg agctcgaatt cgtaatcatg gtcatagctg tttcctgtgtgaaattgtta 1680 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaagcctggggtgc 1740 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctttccagtcggg 1800 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagaggcggtttgcg 1860 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcgttcggctgcg 1920 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaatcaggggataa 1980 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgtaaaaaggccgc 2040 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaaatcgacgctc 2100 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttccccctggaag 2160 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgtccgcctttct 2220 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctcagttcggtgta 2280 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccgaccgctgcgc 2340 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttatcgccactggc 2400 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgctacagagttctt 2460 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatctgcgctctgct 2520 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaacaaaccaccgc 2580 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaaaaggatctca 2640 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaaactcacgtta 2700 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttttaaattaaaa 2760 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgacagttaccaatg 2820 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatccatagttgcctg 2880 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggccccagtgctgc 2940 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataaaccagccagc 3000 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatccagtctattaa 3060 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgcaacgttgttgc 3120 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcattcagctccgg 3180 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaagcggttagctc 3240 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcactcatggttat 3300 ggcagcactg cataattctc ttactgtcat gccatccgta agatgcttttctgtgactgg 3360 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagttgctcttgccc 3420 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgctcatcattgg 3480 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagatccagttcgat 3540 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcaccagcgtttctgg 3600 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcgacacggaaatg 3660 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagggttattgtct 3720 catgagcgga tacatatttg aatgtattta gaaaaataaa caaataggggttccgcgcac 3780 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatgacattaaccta 3840 taaaaatagg cgtatcacga ggccctttcg tc 3872 12 3934 DNA E.coli 12 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccggagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcgtcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgtactgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgcatcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcctcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggtaacgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttcaagccgtcaattgtc 420 tgattcgtta ccaattatga caacttgacg gctacatcat tcactttttcttcacaaccg 480 gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcgagaaatagagt 540 tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggtggtgctcaaa 600 agcagcttcg cctggctgat acgttggtcc tcgcgccagc ttaagacgctaatccctaac 660 tgctggcgga aaagatgtga cagacgcgac ggcgacaagc aaacatgctgtgcgacgctg 720 gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagcctcgcgtacc 780 cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccgcagtaacaat 840 tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttgcccggcgtta 900 atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgggcgaaagaac 960 cccgtattgg caaatattga cggccagtta agccattcat gccagtaggcgcgcggacga 1020 aagtaaaccc actggtgata ccattcgcga gcctccggat gacgaccgtagtgatgaatc 1080 tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcgtccctgattt 1140 ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcattcccagcggt 1200 cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacccgccaccaga 1260 tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagccatacttttc 1320 atactcccgc cattcagaga agaaaccaat tgtccatatt gcatcagacattgccgtcac 1380 tgcgtctttt actggctctt ctcgctaacc aaaccggtaa ccccgcttattaaaagcatt 1440 ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta acaaaagtgtctataatcac 1500 ggcagaaaag tccacattga ttatttgcac ggcgtcacac tttgctatgccatagcattt 1560 ttatccataa gattagcgga tcctacctga cgctttttat cgcaactctctactgtttct 1620 ccatacccgt ttttttgggc tagcaggagg aattcaccct gcaggtcgactctagaggat 1680 ccccgggtac cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgtgtgaaattgt 1740 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaaagcctggggt 1800 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgctttccagtcg 1860 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggagaggcggtttg 1920 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggtcgttcggctg 1980 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacagaatcaggggat 2040 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccgtaaaaaggcc 2100 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaaaaatcgacgc 2160 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtttccccctgga 2220 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacctgtccgccttt 2280 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatctcagttcggtg 2340 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcccgaccgctgc 2400 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgacttatcgccactg 2460 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgctacagagttc 2520 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtatctgcgctctg 2580 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaaacaaaccacc 2640 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaaaaaaggatct 2700 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacgaaaactcacgt 2760 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatccttttaaattaa 2820 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctgacagttaccaa 2880 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatccatagttgcc 2940 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctggccccagtgct 3000 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaataaaccagcca 3060 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccatccagtctatt 3120 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcgcaacgttgtt 3180 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttcattcagctcc 3240 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaaagcggttagc 3300 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatcactcatggtt 3360 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgcttttctgtgact 3420 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgagttgctcttgc 3480 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagtgctcatcatt 3540 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgagatccagttcg 3600 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcaccagcgtttct 3660 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggcgacacggaaa 3720 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatcagggttattgt 3780 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaataggggttccgcgc 3840 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcatgacattaacc 3900 tataaaaata ggcgtatcac gaggcccttt cgtc 3934 13 5953 DNAhomo sapien 13 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatatatggagttccg 60 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacccccgcccatt 120 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttccattgacgtca 180 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgtatcatatgcc 240 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcattatgcccagta 300 catgacctta tgggactttc ctacttggca gtacatctac gtattagtcatcgctattac 360 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttgactcacgggg 420 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcaccaaaatcaacg 480 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcggtaggcgtgt 540 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatccgctagcgcta 600 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcgagggcgagggc 660 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaagctgcccgtg 720 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcagccgctacccc 780 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggctacgtccaggag 840 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggtgaagttcgag 900 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaaggaggacggcaac 960 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatatcatggccgac 1020 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcgaggacggcagc 1080 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccccgtgctgctg 1140 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaacgagaagcgc 1200 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcggcatggacgag 1260 ctgtacaagt ccggactcag atctcgagct taataacaag ccgtcaattgtctgattcgt 1320 taccaattat gacaacttga cggctacatc attcactttt tcttcacaaccggcacggaa 1380 ctcgctcggg ctggccccgg tgcatttttt aaatacccgc gagaaatagagttgatcgtc 1440 aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg gtggtgctcaaaagcagctt 1500 cgcctggctg atacgttggt cctcgcgcca gcttaagacg ctaatccctaactgctggcg 1560 gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgctggcgatatc 1620 aaaattgctg tctgccaggt gatcgctgat gtactgacaa gcctcgcgtacccgattatc 1680 catcggtgga tggagcgact cgttaatcgc ttccatgcgc cgcagtaacaattgctcaag 1740 cagatttatc gccagcagct ccgaatagcg cccttcccct tgcccggcgttaatgatttg 1800 cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc gggcgaaagaaccccgtatt 1860 ggcaaatatt gacggccagt taagccattc atgccagtag gcgcgcggacgaaagtaaac 1920 ccactggtga taccattcgc gagcctccgg atgacgaccg tagtgatgaatctctcctgg 1980 cgggaacagc aaaatatcac ccggtcggca aacaaattct cgtccctgatttttcaccac 2040 cccctgaccg cgaatggtga gattgagaat ataacctttc attcccagcggtcggtcgat 2100 aaaaaaatcg agataaccgt tggcctcaat cggcgttaaa cccgccaccagatgggcatt 2160 aaacgagtat cccggcagca ggggatcatt ttgcgcttca gccatacttttcatactccc 2220 gccattcaga gaagaaacca attgtccata ttgcatcaga cattgccgtcactgcgtctt 2280 ttactggctc ttctcgctaa ccaaaccggt aaccccgctt attaaaagcattctgtaaca 2340 aagcgggacc aaagccatga caaaaacgcg taacaaaagt gtctataatcacggcagaaa 2400 agtccacatt gattatttgc acggcgtcac actttgctat gccatagcatttttatccat 2460 aagattagcg gatcctacct gacgcttttt atcgcaactc tctactgtttctccataccc 2520 gtttttttgg gctagcagga ggaattcacc atggtacccg gggatcctctagagtcgacc 2580 tgcaggcatg caagcttggc ccgcgggccc gggatccacc ggatctagataactgatcat 2640 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcccacacctccc 2700 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgtttattgcagctta 2760 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcatttttttcact 2820 gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtaaattgtaagcg 2880 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattttttaaccaat 2940 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagatagggttgagtg 3000 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaacgtcaaagggc 3060 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaatcaagttttt 3120 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccccgatttagag 3180 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcgaaaggagcgg 3240 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccacacccgccgcgc 3300 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtgcgcggaaccc 3360 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgagacaataaccct 3420 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaagaaccagctgtg 3480 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggcagaagtatgca 3540 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggctccccagcagg 3600 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgcccctaactcc 3660 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatggctgactaat 3720 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattccagaagtagtg 3780 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagacaggatgaggat 3840 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgcttgggtggaga 3900 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgccgccgtgttcc 3960 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtccggtgccctga 4020 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggcgttccttgcg 4080 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattgggcgaagtgc 4140 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatccatcatggctg 4200 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgaccaccaagcga 4260 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgatcaggatgatc 4320 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctcaaggcgagca 4380 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccgaatatcatgg 4440 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtggcggaccgct 4500 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggcgaatgggctg 4560 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatcgccttctatc 4620 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccgaccaagcgac 4680 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaaggttgggctt 4740 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatctcatgctgga 4800 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaataccggaagg 4860 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttgggtcgtttgt 4920 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccaccgagacccca 4980 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaagttcgggtga 5040 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcctcaggttactc 5100 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatctaggtgaagat 5160 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttccactgagcgtc 5220 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgcgcgtaatctg 5280 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccggatcaagagct 5340 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaaatactgtcct 5400 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgcctacatacct 5460 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgtgtcttaccgg 5520 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaacggggggttc 5580 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacctacagcgtga 5640 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatccggtaagcgg 5700 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcctggtatcttta 5760 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgatgctcgtcagg 5820 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcctggccttttg 5880 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtggataaccgtat 5940 taccgccatg cat 5953 14 1380 DNA Rat 14 gaattcaggcgctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcaggt 60 cgacatggcaaccacgcacg cgcagggcca cccgccagtc ttggggaatg atactctccg 120 ggaacattatgattacgtgg ggaagctggc aggcaggctg cgggatcccc ctgagggtag 180 caccctcatcaccaccatcc tcttcttggt cacctgtagc ttcatcgtct tggagaacct 240 gatggttttgattgccatct ggaaaaacaa taaatttcat aaccgcatgt actttttcat 300 cggcaacttggctctctgcg acctgctggc cggcatagcc tacaaggtca acattctgat 360 gtccggtaggaagacgttca gcctgtctcc aacagtgtgg ttcctcaggg agggcagtat 420 gttcgtagccctgggcgcat ccacatgcag cttattggcc attgccattg agcggcacct 480 gaccatgatcaagatgaggc cgtacgacgc caacaagaag caccgcgtgt tccttctgat 540 tgggatgtgctggctaattg ccttctcgct gggtgccctg cccatcctgg gctggaactg 600 cctggaaaactttcccgact gctctaccat cttgcccctc tactccaaga aatacattgc 660 ctttctcatcagcatcttca tagccattct ggtgaccatc gtcatcttgt acgcgcgcat 720 ctacttcctggtcaagtcca gcagccgcag ggtggccaac cacaactccg agagatccat 780 ggcccttctgcggaccgtag tgatcgtggt gagcgtgttc atcgcctgtt ggtcccccct 840 tttcatcctcttcctcatcg atgtggcctg cagggcgaag gagtgctcca tcctcttcaa 900 gagtcagtggttcatcatgc tggctgtcct caactcggcc atgaaccctg tcatctacac 960 gctggccagcaaagagatgc ggcgtgcttt cttccggttg gtgtgcggct gtctggtcaa 1020 gggcaaggggacccaggcct ccccgatgca gcctgctctt gacccgagca gaagtaaatc 1080 aagctccagtaacaacagca gcagccactc tccaaaggtc aaggaagacc tgccccatgt 1140 ggctacctcttcctgcgtta ctgacaaaac gaggtcgctt cagaatgggg tcctctgcaa 1200 gaagggcaattctgcagata tccagcacag tggcggccgc tcgagtctag agggcccgcg 1260 gttcgaaggtaagcctatcc ctaaccctct cctcggtctc gattctacgc gtaccggtca 1320 tcatcaccatcaccattgat aaggtaccga gctcgaattc gtaatcatgg tcatagctgt 1380 15 1320 DNAArtificial Sequence Fusion protein 15 gaattcaggc gctttttaga ctggtcgtaatgaaattcag caggatcaca ttctgcaggt 60 cgacatgggg caacccggga acggcagcgccttcttgctg gcacccaatg gaagccatgc 120 gccggaccac gacgtcacgc agcaaagggacgaggtgtgg gtggtgggca tgggcatcgt 180 catgtctctc atcgtcctgg ccatcgtgtttggcaatgtg ctggtcatca cagccattgc 240 caagttcgag cgtctgcaga cggtcaccaactacttcatc acttcactgg cctgtgctga 300 tctggtcatg ggcctagcag tggtgccctttggggccgcc catattctta tgaaaatgtg 360 gacttttggc aacttctggt gcgagttttggacttccatt gatgtgctgt gcgtcacggc 420 cagcattgag accctgtgcg tgatcgcagtggatcgctac tttgccatta cttcaccttt 480 caagtaccag agcctgctga ccaagaataaggcccgggtg atcattctga tggtgtggat 540 tgtgtcaggc cttayctcct tcttgcccattcagatgcac tggtacaggg ccacccacca 600 ggaagccatc aactgctatg ccaatgagacctgctgtgac ttcttcacga accaagccta 660 tgccattgcc tcttccatcg tgtccttctacgttcccctg gtgatcatgg tcttcgtcta 720 ctccagggtc tttcaggagg ccaaaaggcagctccagaag attgacaaat ctgagggccg 780 cttccatgtc cagaacctta gccaggtggagcaggatggg cggacggggc atggactccg 840 cagatcttcc aagttctgct tgaaggagcacaaagccctc aagacgttag gcatcatcat 900 gggcactttc accctctgct ggctgcccttcttcatcgtt aacattgtgc atgtgatcca 960 ggataacctc atccgtaagg aagtttacatcctcctaaat tggataggct atgtcaattc 1020 tggtttcaat ccccttatct actgccggagcccagatttc aggattgcct tccaggagct 1080 tctgtgcctg cgcaggtctt ctttgaaggcctatggcaat ggctactcca gcaacggcaa 1140 cacaggggag cagagtggat atcacgtggaacaggagaaa gaaaataaac tgctgtgtga 1200 agacctccca ggcacggaag actttgtgggccatcaaggt actgtgccta gcgataacat 1260 tgattcacaa gggaggaatt gtagtacaaatgactcactg ctataataag gatccccggg 1320 16 52 DNA Artificial SequenceCloning primer 16 aattggtacc tcaatgatga tgatgatgat gcttgcagag gaccccattctg 52 17 1320 DNA Artificial Sequence Fusion protein 17 tcgcaactctctactgtttc tccatacccg tttttttggg ctagcaggag gaattcacca 60 tggatagtgtgtgtccccaa ggaaaatata tccaccctca aaataattcg atttgctgta 120 ccaagtgccacaaaggaacc tacttgtaca atgactgtcc aggcccgggg caggatacgg 180 actgcagggagtgtgagagc ggctccttca ccgcttcaga aaaccacctc agacactgcc 240 tcagctgctccaaatgccga aaggaaatgg gtcaggtgga gatctcttct tgcacagtgg 300 accgggacaccgtgtgtggc tgcaggaaga accagtaccg gcattattgg agtgaaaacc 360 ttttccagtgcttcaattgc agcctctgcc tcaatgggac cgtgcacctc tcctgccagg 420 agaaacagaacaccgtgtgc acctgccatg caggtttctt tctaagagaa aacgagtgtg 480 tctcctgtagtaactgtaag aaaagcctgg agtgcacgaa gttgtgccta ccccagattg 540 agaatgttaagggcactgag gactcaggca ccacagtgct gttgcccctg gtcattttct 600 ttggtctttgccttttatcc ctcctcttca ttggtttaat gtatcgctac caacggtgga 660 agtccaagctctactccatt gtttgtggga aatcgacacc tgaaaaagag ggggagcttg 720 aaggaactactactaagccc ctggccccaa acccaagctt cagtcccact ccaggcttca 780 cccccaccctgggcttcagt cccgtgccca gttccacctt cacctccagc tccacctata 840 cccccggtgactgtcccaac tttgcggctc cccgcagaga ggtggcacca ccctatcagg 900 gggctgaccccatccttgcg acagccctcg cctccgaccc catccccaac ccccttcaga 960 agtgggaggacagcgcccac aagccacaga gcctagacac tgatgacccc gcgacgctgt 1020 acgccgtggtggagaacgtg cccccgttgc gctggaagga attcgtgcgg cgcctagggc 1080 tgagcgaccacgagatcgat cggctggagc tgcagaacgg gcgctgcctg cgcgaggcgc 1140 aatacagcatgctggcgacc tggaggcggc gcacgccgcg gcgcgaggcc acgctggagc 1200 tgctgggacgcgtgctccgc gacatggacc tgctgggctg cctggaggac atcgaggagg 1260 cgctttgcggccccgccgcc ctcccgcccg cgcccagtct tctcagatga tctagagtcg 1320 18 1380 DNAArtificial Sequence Fusion protein 18 ccatacccgt ttttttgggc tagcaggaggaattcaccct gcaggtcgac atgggactgg 60 tccctcacct aggggacagg gagaagagagatagtgtgtg tccccaagga aaatatatcc 120 accctcaaaa taattcgatt tgctgtaccaagtgccacaa aggaacctac ttgtacaatg 180 actgtccagg cccggggcag gatacggactgcagggagtg tgagagcggc tccttcaccg 240 cttcagaaaa ccacctcaga cactgcctcagctgctccaa atgccgaaag gaaatgggtc 300 aggtggagat ctcttcttgc acagtggaccgggacaccgt gtgtggctgc aggaagaacc 360 agtaccggca ttattggagt gaaaaccttttccagtgctt caattgcagc ctctgcctca 420 atgggaccgt gcacctctcc tgccaggagaaacagaacac cgtgtgcacc tgccatgcag 480 gtttctttct aagagaaaac gagtgtgtctcctgtagtaa ctgtaagaaa agcctggagt 540 gcacgaagtt gtgcctaccc cagattgagaatgttaaggg cactgaggac tcaggcacca 600 cagtgctgtt gcccctggtc attttctttggtctttgcct tttatccctc ctcttcattg 660 gtttaatgta tcgctaccaa cggtggaagtccaagctcta ctccattgtt tgtgggaaat 720 cgacacctga aaaagagggg gagcttgaaggaactactac taagcccctg gccccaaacc 780 caagcttcag tcccactcca ggcttcacccccaccctggg cttcagtccc gtgcccagtt 840 ccaccttcac ctccagctcc acctatacccccggtgactg tcccaacttt gcggctcccc 900 gcagagaggt ggcaccaccc tatcagggggctgaccccat ccttgcgaca gccctcgcct 960 ccgaccccat ccccaacccc cttcagaagtgggaggacag cgcccacaag ccacagagcc 1020 tagacactga tgaccccgcg acgctgtacgccgtggtgga gaacgtgccc ccgttgcgct 1080 ggaaggaatt cgtgcggcgc ctagggctgagcgaccacga gatcgatcgg ctggagctgc 1140 agaacgggcg ctgcctgcgc gaggcgcaatacagcatgct ggcgacctgg aggcggcgca 1200 cgccgcggcg cgaggccacg ctggagctgctgggacgcgt gctccgcgac atggacctgc 1260 tgggctgcct ggaggacatc gaggaggcgctttgcggccc cgccgccctc ccgcccgcgc 1320 ccagtcttct cagataataa ggtaccgagctcgaattcgt aatcatggtc atagctgttt 1380 19 780 DNA Artificial SequenceFusion protein 19 gtttttttgg gctagcagga ggaattcatg agcactgaaa gcatgatccgggacgtggag 60 ctggccgagg aggcgctccc caagaagaca ggggggcccc agggctccaggcggtgcttg 120 ttcctcagcc tcttctcctt cctgatcgtg gcaggcgcca ccacgctcttctgcctgctg 180 cactttggag tgatcggccc ccagagggaa gagttcccca gggacctctctctaatcagc 240 cctctggccc aggcagtcag atcatcttct cgaaccccga gtgacaagcctgtagcccat 300 gttgtagcaa accctcaagc tgaggggcag ctccagtggc tgaaccgccgggccaatgcc 360 ctcctggcca atggcgtgga gctgagagat aaccagctgg tggtgccatcagagggcctg 420 tacctcatct actcccaggt cctcttcaag ggccaaggct gcccctccacccatgtgctc 480 ctcacccaca ccatcagccg catcgccgtc tcctaccaga ccaaggtcaacctcctctct 540 gccatcaaga gcccctgcca gagggagacc ccagaggggg ctgaggccaagccctggtat 600 gagcccatct atctgggagg ggtcttccag ctggagaagg gtgaccgactcagcgctgag 660 atcaatcggc ccgactatct cgactttgcc gagtctgggc aggtctactttgggatcatt 720 gccctgtgat aagcttggcc cgcgggcccg ggatccaccg gatctagataactgatcata 780 20 300 DNA Artificial Sequence Fusion protein 20gtttttttgg gctagcagga ggaattcacc atggtaccat gaacttgggg aatcgactgt 60ttattctgat agcggtctta cttcccctcg cagtattact gctcaatagt gactctgaat 120gtcccctgtc ccacgatggg tactgcctcc atgatggtgt gtgcatgtat attgaagcat 180tggacaagta tgcatgcaac tgtgttgttg gctacatcgg ggagcgatgt cagtaccgag 240acctgaagtg gtgggaactg cgctaataag cttggcccgc gggcccggga tccaccggat 300 211620 DNA Artificial Sequence Fusion protein 21 gtttttttgg gctagcaggaggaattcacc atgaacttgg ggaatcgact gtttattctg 60 atagcggtct tacttcccctcgcagtatta ctgctctcat tcacattgag cgtcaccgtt 120 cagcagcctc agttgacattaacggcggcc gtcattggtg atggcgcacc ggctaatggg 180 aaaactgcaa tcaccgttgagttcaccgtt gctgattttg aggggaaacc cttagccggg 240 caggaggtgg tgataaccaccaataatggt gcgctaccga ataaaatcac ggaaaagaca 300 gatgcaaatg gcgtcgcgcgcattgcatta accaatacga cagatggcgt gacggtagtc 360 acagcagaag tggaggggcaacggcaaagt gttgataccc actttgttaa gggtactatc 420 gcggcggata aatccactctggctgcggta ccgacatcta tcatcgctga tggtctaatg 480 gcttcaacca tcacgttggagttgaaggat acctatgggg acccgcaggc tggcgcgaat 540 gtggcttttg acacaaccttaggcaatatg ggcgttatca cggatcacaa tgacggcact 600 tatagcgcac cattgaccagtaccacgttg ggggtagcaa cagtaacggt gaaagtggat 660 ggggctgcgt tcagtgtgccgagtgtgacg gttaatttca cggcagatcc tattccagat 720 gctggccgct ccagtttcaccgtctccaca ccggatatct tggctgatgg cacgatgagt 780 tccacattat cctttgtccctgtcgataag aatggccatt ttatcagtgg gatgcagggc 840 ttgagtttta ctcaaaacggtgtgccggtg agtattagcc ccattaccga gcagccagat 900 agctataccg cgacggtggttgggaatagt gtcggtgatg tcacaatcac gccgcaggtt 960 gataccctga tactgagtacattgcagaaa aaaatatccc tattcccggt acctacgctg 1020 accggtattc tggttaacgggcaaaatttc gctacggata aagggttccc gaaaacgatc 1080 tttaaaaacg ccacattccagttacagatg gataacgatg ttgctaataa tactcagtat 1140 gagtggtcgt cgtcattcacacccaatgta tcggttaacg atcagggtca ggtgacgatt 1200 acctaccaaa cctatagcgaagtggctgtg acggcgaaaa gtaaaaaatt cccaagttat 1260 tcggtgagtt atcggttctacccaaatcgg tggatatacg atggcggcag atcgctggta 1320 tccagtctcg aggccagcagacaatgccaa ggttcagata tgtctgcggt tcttgaatcc 1380 tcacgtgcaa ccaacggaacgcgtgcgcct gacgggacat tgtggggcga gtgggggagc 1440 ttgaccgcgt atagttctgattggcaatct ggtgaatatt gggtcaaaaa gaccagcacg 1500 gattttgaaa ccatgaatatggacacaggc gcactgcaac cagggcctgc atacttggcg 1560 ttcccgctct gtgcgctgtcaatataactg caggcatgca agcttggccc gcgggcccgg 1620 22 208 DNA ArtificialSequence Fusion protein 22 gaattcaggc gctttttaga ctggtcgtaa tgaaattcagcaggatcaca ttctgcagat 60 gtcacggccg agacttatag tcgctttgtt tttattttttaatgtatttg tacatggaga 120 aaataaagtg aaacaaagca ctattgcact ggcactcttaccgttactgt ttacccctgt 180 gacaaaagcc cggacaccag aatctaga 208 23 1546 DNAArtificial Sequence Fusion protein 23 gaattcaggc gctttttaga ctggtcgtaatgaaattcag caggatcaca ttctgcagat 60 gtcacggccg agacttatag tcgctttgtttttatttttt aatgtatttg tacatggaga 120 aaataaagtg aaacaaagca ctattgcactggcactctta ccgttactgt ttacccctgt 180 gacaaaagcc cggacaccag aaatgcctgttctggaaaac cgggctgctc agggcgatat 240 tactgcaccc ggcggtgctc gccgtttaacgggtgatcag actgccgctc tgcgtgattc 300 tcttagcgat aaacctgcaa aaaatattattttgctgatt ggcgatggga tgggggactc 360 ggaaattact gccgcacgta attatgccgaaggtgcgggc ggctttttta aaggtataga 420 tgccttaccg cttaccgggc aatacactcactatgcgctg aataaaaaaa ccggcaaacc 480 ggactacgtc accgactcgg ctgcatcagcaaccgcctgg tcaaccggtg tcaaaaccta 540 taacggcgcg ctgggcgtcg atattcacgaaaaagatcac ccaacgattc tggaaatggc 600 aaaagccgca ggtctggcga ccggtaacgtttctaccgca gagttgcagg atgccacgcc 660 cgctgcgctg gtggcacatg tgacctcgcgcaaatgctac ggtccgagcg cgaccagtga 720 aaaatgtccg ggtaacgctc tggaaaaaggcggaaaagga tcgattaccg aacagctgct 780 taacgctcgt gccgacgtta cgcttggcggcggcgcaaaa acctttgctg aaacggcaac 840 cgctggtgaa tggcagggaa aaacgctgcgtgaacaggca caggcgcgtg gttatcagtt 900 ggtgagcgat gctgcctcac tgaattcggtgacggaagcg aatcagcaaa aacccctgct 960 tggcctgttt gctgacggca atatgccagtgcgctggcta ggaccgaaag caacgtacca 1020 tggcaatatc gataagcccg cagtcacctgtacgccaaat ccgcaacgta atgacagtgt 1080 accaaccctg gcgcagatga ccgacaaagccattgaattg ttgagtaaaa atgagaaagg 1140 ctttttcctg caagttgaag gtgcgtcaatcgataaacag gatcatgctg cgaatccttg 1200 tgggcaaatt ggcgagacgg tcgatctcgatgaagccgta caacgggcgc tggaattcgc 1260 taaaaaggag ggtaacacgc tggtcatagtcaccgctgat cacgcccacg ccagccagat 1320 tgttgcgccg gataccaaag ctccgggcctcacccaggcg ctaaatacca aagatggcgc 1380 agtgatggtg atgagttacg ggaactccgaagaggattca caagaacata ccggcagtca 1440 gttgcgtatt gcggcgtatg gcccgcatgccgccaatgtt gttggactga ccgaccagac 1500 cgatctcttc tacaccatga aagccgctctggggctgaaa tctaga 1546 24 148 DNA Artificial Sequence Fusion protein 24gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcagat 60gaaaataaaa acaggtgcac gcatcctcgc attatccgca ttaacgacga tgatgttttc 120cgcctcggct ctcgccaaaa tctctaga 148 25 1174 DNA Artificial SequenceFusion protein 25 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caggatcacattctgcagat 60 gaaaataaaa acaggtgcac gcatcctcgc attatccgca ttaacgacgatgatgttttc 120 cgcctcggct ctcgccaaaa tcgaagaagg taaactggta atctggattaacggcgataa 180 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccggaattaaagt 240 caccgttgag catccggata aactggaaga gaaattccca caggttgcggcaactggcga 300 tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctcaatctggcct 360 gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgtttacctggga 420 tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaagcgttatcgct 480 gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagagatcccggcgct 540 ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgcaagaaccgta 600 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatgaaaacggcaa 660 gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtctgaccttcct 720 ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactccatcgcagaagc 780 tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcatggtccaacat 840 cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagggtcaaccatc 900 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccgaacaaagagct 960 ggcgaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaagcggttaataa 1020 agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttggcgaaagatcc 1080 acgtattgcc gccaccatgg aaaacgccca gaaaggtgaa atcatgccgaacatcccgca 1140 gatgtccgct ttctggtatg ccgtgcgttc taga 1174 26 3840 DNAArtificial Sequence Fusion protein 26 accatatgcg gtgtgaaata ccgcacagatgcgtaaggag aaaataccgc atcaggcgcc 60 atacgcgaca gcgcgcaata accgttctcgactcataaaa gtgatgccgc tataatgccg 120 cgtcctattt gaatgctttc gggatgattctggtaacagg gaatgtgatt gattataaga 180 acatcccggt tccgcgaagc caacaacctgtgcttgcggg gtaagagttg accgagcact 240 gtgatttttt gaggtaacaa gatgcaagtttcagttgaaa ccactcaagg ccttggccgc 300 cgtgtaacga ttactatcgc tgctgacagcatcgagaccg ctgttaaaag cgagctggtc 360 aacgttgcga aaaaagtacg tattgacggcttccgcaaag gcaaagtgcc aatgaatatc 420 gttgctcagc gttatggcgc gtctgtacgccaggacgttc tgggtgacct gatgagccgt 480 aacttcattg acgccatcat taaagaaaaaatcaatccgg ctggcgcacc gacttatgtt 540 ccgggcgaat acaagctggg tgaagacttcacttactctg tagagtttga agtttatccg 600 gaagttgaac tgcagggtct ggaagcgatcgaagttgaaa aaccgatcgt tgaagtgacc 660 gacgctgacg ttgacggcat gctggatactctgcgtaaac agcaggcgac ctggaaagaa 720 aaagacggcg ctgttgaagc agaagaccgcgtaaccatcg acttcaccgg ttctgtagac 780 ggcgaagagt tcgaaggcgg taaagcgtctgatttcgtac tggcgatggg ccagggtcgt 840 atgatcccgg gctttgaaga cggtatcaaaggccacaaag ctggcgaaga gttcaccatc 900 gacgtgacct tcccggaaga ataccacgcagaaaacctga aaggtaaagc agcgaaattc 960 gctatcaacc tgaagaaagt tgaagagcgtgaactgccgg aactgactgc agaattcatc 1020 aaacgtttcg gcgttgaaga tggttccgtagaaggtctgc gcgctgaagt gcgtaaaaac 1080 atggagcgcg agctgaagag cgccatccgtaaccgcgtta agtctcaggc gatcgaaggt 1140 ctggtaaaag ctaacgacat cgacgtaccggctgcgctga tcgacagcga aatcgacgtt 1200 ctgcgtcgcc aggctgcaca gcgtttcggtggcaacgaaa aacaagctct ggaactgccg 1260 cgcgaactgt tcgaagaaca ggctaaacgccgcgtagttg ttggcctgct gctgggcgaa 1320 gttatccgca ccaacgagct gaaagctgacgaagagcgcg tgaaaggcct gatcgaagag 1380 atggcttctg cgtacgaaga tccgaaagaagttatcgagt tctacagcaa aaacaaagaa 1440 ctgatggaca acatgcgcaa tgttgctctggaagaacagg ctgttgaagc tgtactggcg 1500 aaagcgaaag tgactgaaaa agaaaccactttcaacgagc tgatgaacca gcaggcgtaa 1560 taataatcta gaggtagcac aatcagattcgcttatgacg gcgatgaaga aattgcgatg 1620 aaatgtgagg tgaatcaggg ttttcacccgattttgtgct gatcagaatt ttttttcttt 1680 ttcccccttg aaggggcgaa gcctcatccccatttctctg gtcaccagcc gggaaaccac 1740 gtaagctccg gcgtcaccca taacagatacggactttctc aaaggagagt tatcaatgaa 1800 tattcgtcca ttgcatgatc gcgtgatcgtcaagcgtaaa gaagttgaaa ctaaatctgc 1860 tggcggcatc gttctgaccg gctctgcagcggctaaatcc acccgcggcg aagtgctggc 1920 tgtcggcaat ggccgtatcc ttgaaaatggcgaagtgaag ccgctggatg tgaaagttgg 1980 cgacatcgtt attttcaacg atggctacggtgtgaaatct gagaagatcg acaatgaaga 2040 agtgttgatc atgtccgaaa gcgacattctggcaattgtt gaagcgtaat ccgcgcacga 2100 cactgaacat acgaatttaa ggaataaagataatggcagc taaagacgta aaattcggta 2160 acgacgctcg tgtgaaaatg ctgcgcggcgtaaacgtact ggcagatgca gtgaaagtta 2220 ccctcggtcc aaaaggccgt aacgtagttctggataaatc tttcggtgca ccgaccatca 2280 ccaaagatgg tgtttccgtt gctcgtgaaatcgaactgga agacaagttc gaaaatatgg 2340 gtgcgcagat ggtgaaagaa gttgcctctaaagcaaacga cgctgcaggc gacggtacca 2400 ccactgcaac cgtactggct caggctatcatcactgaagg tctgaaagct gttgctgcgg 2460 gcatgaaccc gatggacctg aaacgtggtatcgacaaagc ggttaccgct gcagttgaag 2520 aactgaaagc gctgtccgta ccatgctctgactctaaagc gattgctcag gttggtacca 2580 tctccgctaa ctccgacgaa accgtaggtaaactgatcgc tgaagcgatg gacaaagtcg 2640 gtaaagaagg cgttatcacc gttgaagacggtaccggtct gcaggacgaa ctggacgtgg 2700 ttgaaggtat gcagttcgac cgtggctacctgtctcctta cttcatcaac aagccggaaa 2760 ctggcgcagt agaactggaa agcccgttcatcctgctggc tgacaagaaa atctccaaca 2820 tccgcgaaat gctgccggtt ctggaagctgttgccaaagc aggcaaaccg ctgctgatca 2880 tcgctgaaga tgtagaaggc gaagcgctggcaactctggt tgttaacacc atgcgtggca 2940 tcgtgaaagt cgctgcggtt aaagcaccgggcttcggcga tcgtcgtaaa gctatgctgc 3000 aggatatcgc aaccctgact ggcggtaccgtgatctctga agagatcggt atggagctgg 3060 aaaaagcaac cctggaagac ctgggtcaggctaaacgtgt tgtgatcaac aaagacacca 3120 ccactatcat cgatggcgtg ggtgaagaagctgcaatcca gggccgtgtt gctcagatcc 3180 gtcagcagat tgaagaagca acttctgactacgaccgtga aaaactgcag gaacgcgtag 3240 cgaaactggc aggcggcgtt gcagttatcaaagtgggtgc tgctaccgaa gttgaaatga 3300 aagagaaaaa agcacgcgtt gaagatgccctgcacgcgac ccgtgctgcg gtagaagaag 3360 gcgtggttgc tggtggtggt gttgcgctgatccgcgtagc gtctaaactg gctgacctgc 3420 gtggtcagaa cgaagaccag aacgtgggtatcaaagttgc actgcgtgca atggaagctc 3480 cgctgcgtca gatcgtattg aactgcggcgaagaaccgtc tgttgttgct aacaccgtta 3540 aaggcggcga cggcaactac ggttacaacgcagcaaccga agaatacggc aacatgatcg 3600 acatgggtat cctggatcca accaaagtaactcgttctgc tctgcagtac gcagcttctg 3660 tggctggcct gatgatcacc accgaatgcatggttaccga cctgccgaaa aacgatgcag 3720 ctgacttagg cgctgctggc ggtatgggcggcatgggtgg catgggcggc atgatgtaat 3780 aataagcttg catgcctgca ggtcgactctagaggatccc cgggtaccga gctcgaattc 3840 27 480 DNA Rat Cloning primer 27gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcagat 60gatcgaagcc cgctctagac tcgagagcga taaaattatt cacctgactg acgacagttt 120tgacacggat gtactcaaag cggacggggc gatcctcgtc gatttctggg cagagtggtg 180cggtccgtgc aaaatgatcg ccccgattct ggatgaaatc gctgacgaat atcagggcaa 240actgaccgtt gcaaaactga acatcgatca aaaccctggc actgcgccga aatatggcat 300ccgtggtatc ccgactctgc tgctgttcaa aaacggtgaa gtggcggcaa ccaaagtggg 360tgcactgtct aaaggtcagt tgaaagagtt cctcgacgct aacctggcgc tcgaggatta 420taaagatcat gatggcgatt ataaagatca tgatgattaa taaggatccc cgggtaccga 480 281140 DNA Rat Cloning primer 28 atggcaacca cgcacgcgca ggggcacccgccagtcttgg ggaatgatac tctccgggaa 60 cattatgatt acgtggggaa gctggcaggcaggctgcggg atccccctga gggtagcacc 120 ctcatcacca ccatcctctt cttggtcacctgtagcttca tcgtcttgga gaacctgatg 180 gttttgattg ccatctggaa aaacaataaatttcataacc gcatgtactt tttcatcggc 240 aacttggctc tctgcgacct gctggccggcatagcctaca aggtcaacat tctgatgtcc 300 ggtaggaaga cgttcagcct gtctccaacagtgtggttcc tcagggaggg cagtatgttc 360 gtagccctgg gcgcatccac atgcagcttattggccattg ccattgagcg gcacctgacc 420 atgatcaaga tgaggccgta cgacgccaacaagaagcacc gcgtgttcct tctgattggg 480 atgtgctggc taattgcctt ctcgctgggtgccctgccca tcctgggctg gaactgcctg 540 gagaactttc ccgactgctc taccatcttgcccctctact ccaagaaata cattgccttt 600 ctcatcagca tcttcacagc cattctggtgaccatcgtca tcttgtacgc gcgcatctac 660 ttcctggtca agtccagcag ccgcagggtggccaaccaca actccgagag atccatggcc 720 cttctgcgga ccgtagtgat cgtggtgagcgtgttcatcg cctgttggtc cccccttttc 780 atcctcttcc tcatcgatgt ggcctgcagggcgaaggagt gctccatcct cttcaagagt 840 cagtggttca tcatgctggc tgtcctcaactcggccatga accctgtcat ctacacgctg 900 gccagcaaag agatgcggcg tgctttcttccggttggtgt gcggctgtct ggtcaagggc 960 aaggggaccc aggcctcccc gatgcagcctgctcttgacc cgagcagaag taaatcaagc 1020 tccagtaaca acagcagcag ccactctccaaaggtcaagg aagacctgcc ccatgtggct 1080 acctcttcct gcgtcactga caaaacgaggtcgcttcaga atggggtcct ctgcaagtga 1140 29 379 PRT Rat Cloning primer 29Met Ala Thr Thr His Ala Gln Gly His Pro Pro Val Leu Gly Asn Asp 1 5 1015 Thr Leu Arg Glu His Tyr Asp Tyr Val Gly Lys Leu Ala Gly Arg Leu 20 2530 Arg Asp Pro Pro Glu Gly Ser Thr Leu Ile Thr Thr Ile Leu Phe Leu 35 4045 Val Thr Cys Ser Phe Ile Val Leu Glu Asn Leu Met Val Leu Ile Ala 50 5560 Ile Trp Lys Asn Asn Lys Phe His Asn Arg Met Tyr Phe Phe Ile Gly 65 7075 80 Asn Leu Ala Leu Cys Asp Leu Leu Ala Gly Ile Ala Tyr Lys Val Asn 8590 95 Ile Leu Met Ser Gly Arg Lys Thr Phe Ser Leu Ser Pro Thr Val Trp100 105 110 Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu Gly Ala Ser ThrCys 115 120 125 Ser Leu Leu Ala Ile Ala Ile Glu Arg His Leu Thr Met IleLys Met 130 135 140 Arg Pro Tyr Asp Ala Asn Lys Lys His Arg Val Phe LeuLeu Ile Gly 145 150 155 160 Met Cys Trp Leu Ile Ala Phe Ser Leu Gly AlaLeu Pro Ile Leu Gly 165 170 175 Trp Asn Cys Leu Glu Asn Phe Pro Asp CysSer Thr Ile Leu Pro Leu 180 185 190 Tyr Ser Lys Lys Tyr Ile Ala Phe LeuIle Ser Ile Phe Thr Ala Ile 195 200 205 Leu Val Thr Ile Val Ile Leu TyrAla Arg Ile Tyr Phe Leu Val Lys 210 215 220 Ser Ser Ser Arg Arg Val AlaAsn His Asn Ser Glu Arg Ser Met Ala 225 230 235 240 Leu Leu Arg Thr ValVal Ile Val Val Ser Val Phe Ile Ala Cys Trp 245 250 255 Ser Pro Leu PheIle Leu Phe Leu Ile Asp Val Ala Cys Arg Ala Lys 260 265 270 Glu Cys SerIle Leu Phe Lys Ser Gln Trp Phe Ile Met Leu Ala Val 275 280 285 Leu AsnSer Ala Met Asn Pro Val Ile Tyr Thr Leu Ala Ser Lys Glu 290 295 300 MetArg Arg Ala Phe Phe Arg Leu Val Cys Gly Cys Leu Val Lys Gly 305 310 315320 Lys Gly Thr Gln Ala Ser Pro Met Gln Pro Ala Leu Asp Pro Ser Arg 325330 335 Ser Lys Ser Ser Ser Ser Asn Asn Ser Ser Ser His Ser Pro Lys Val340 345 350 Lys Glu Asp Leu Pro His Val Ala Thr Ser Ser Cys Val Thr AspLys 355 360 365 Thr Arg Ser Leu Gln Asn Gly Val Leu Cys Lys 370 375 3032 DNA Artificial Sequence Cloning primer 30 aattgctagc tccaccagcatcccagtggt ta 32 31 32 DNA Artificial Sequence Cloning primer 31aattggatcc ttaagaagaa gaattgacgt tt 32 32 32 DNA Artificial SequenceCloning primer 32 aattggatcc agaagaagaa ttgacgtttc ca 32 33 52 DNAArtificial Sequence Cloning primer 33 aattggatcc ttaatgatga tgatgatgatgagaagaaga attgacgttt cc 52 34 24 DNA Artificial Sequence Cloning primer34 ttatggcaac cacgcacgcg cagg 24 35 21 DNA Artificial Sequence Cloningprimer 35 agaccgtcac ttgcagagga c 21 36 32 DNA Artificial SequenceCloning primer 36 aattgctagc acgcacgcgc aggggcaccc gc 32 37 34 DNAArtificial Sequence Cloning primer 37 aattggtacc tcacttgcag aggaccccattctg 34 38 32 DNA Artificial Sequence Cloning primer 38 aattgctagcacgcacgcgc aggggcaccc gc 32 39 21 DNA Artificial Sequence Cloning primer39 ggtcgccacc atggtgagca a 21 40 31 DNA Artificial Sequence Cloningprimer 40 ttaaggatcc ttacttgtac agctcgtcca t 31 41 21 DNA ArtificialSequence Cloning primer 41 ggtcgccacc atggtgagca a 21 42 31 DNAArtificial Sequence Cloning primer 42 ttaaggatcc cttgtacagc tcgtccatgc c31 43 25 DNA Artificial Sequence Cloning primer 43 ccaatggaac ttaccaatgacgcgg 25 44 24 DNA Artificial Sequence Cloning primer 44 gcttgcttacgcaggaatgc tggg 24 45 45 DNA Artificial Sequence Cloning primer 45cgcggctgca gatgtttgaa ccaatggaac ttaccaatga cgcgg 45 46 45 DNAArtificial Sequence Cloning primer 46 gcgcctctag attattaatc agcttgcttacgcaggaatg ctggg 45 47 31 DNA Artificial Sequence Cloning primer 47gctagactgg gcggttttat ggacagcaag c 31 48 35 DNA Artificial SequenceCloning primer 48 gcgttaataa ttcagaagaa ctcgtcaaga aggcg 35 49 99 DNAArtificial Sequence Cloning primer 49 gcgcctactg acgtagttcg accgtcggactagcgaagtt cctatacttt ctagagaata 60 ggaacttcgc tagactgggc ggttttatggacagcaagc 99 50 109 DNA Artificial Sequence Cloning primer 50 caagatgctttgcctttgtc tgagttgata ctggctttgg gaagttccta ttctctagaa 60 agtataggaacttcgcgtta ataattcaga agaactcgtc aagaaggcg 109 51 26 DNA ArtificialSequence Cloning primer 51 cgttaccaat tatgacaact tgacgg 26 52 29 DNAArtificial Sequence Cloning primer 52 ttaatctttc tgcgaattga gatgacgcc 2953 28 DNA Artificial Sequence Cloning primer 53 gtgagtcgat attgtctttgttgaccag 28 54 66 DNA Artificial Sequence Cloning primer 54 gcctgcattgcggcgcttca gtctccgctg catactgtcc cgttaccaat tatgacaact 60 tgacgg 66 5569 DNA Artificial Sequence Cloning primer 55 gcctgcattg cggcgcttcagtctccgctg catactgtcc ttaatctttc tgcgaattga 60 gatgacgcc 69 56 76 DNAArtificial Sequence Cloning primer 56 gcctgcattg cggcgcttca gtctccgctgcatactgtcc ttaataaagt gagtcgatat 60 tgtctttgtt gaccag 76 57 66 DNAArtificial Sequence Cloning primer 57 gcctgcattg cggcgcttca gtctccgctgcatactgtcc cgttaccaat tatgacaact 60 tgacgg 66 58 30 DNA ArtificialSequence Cloning primer 58 gcctgttctg gaaaaccggg ctgctcaggg 30 59 28 DNAArtificial Sequence Cloning primer 59 gcggctttca tggtgtagaa gagatcgg 2860 43 DNA Artificial Sequence Cloning primer 60 ccgcgctgca gatgcctgttctggaaaacc gggctgctca ggg 43 61 58 DNA Artificial Sequence Cloningprimer 61 gcgcctctag attattattt cagccccaga gcggctttca tggtgtagaagagatcgg 58 62 24 DNA Artificial Sequence Cloning primer 62 gtcacggccgagacttatag tcgc 24 63 28 DNA Artificial Sequence Cloning primer 63gcggctttca tggtgtagaa gagatcgg 28 64 37 DNA Artificial Sequence Cloningprimer 64 ccgcgctgca gatgtcacgg ccgagactta tagtcgc 37 65 58 DNAArtificial Sequence Cloning primer 65 gcgcctctag attattattt cagccccagagcggctttca tggtgtagaa gagatcgg 58 66 109 DNA Artificial Sequence Cloningprimer 66 ccgcgctgca gatgaacttg gggaatcgac tgtttattct gatagcggtcttacttcccc 60 tcgcagtatt actgctcatg cctgttctgg aaaaccgggc tgctcaggg 10967 58 DNA Artificial Sequence Cloning primer 67 gcgcctctag attattatttcagccccaga gcggctttca tggtgtagaa gagatcgg 58 68 24 DNA ArtificialSequence Cloning primer 68 gcgaattgag atgacgccac tggc 24 69 25 DNAArtificial Sequence Cloning primer 69 cctgctgaat ttcattaacg accag 25 7048 DNA Artificial Sequence Cloning primer 70 cggcgaagct taattaatctttctgcgaat tgagatgacg ccactggc 48 71 53 DNA Artificial Sequence Cloningprimer 71 cgccgtaatc gccgctgcag aatgtgatcc tgctgaattt cattaacgac cag 5372 22 DNA Artificial Sequence Cloning primer 72 cgcagcgctg ttcctttgct cg22 73 23 DNA Artificial Sequence Cloning primer 73 cctcattaag ataataatactgg 23 74 33 DNA Artificial Sequence Cloning primer 74 gccgcaagcttcgcagcgct gttcctttgc tcg 33 75 47 DNA Artificial Sequence Cloningprimer 75 ccaatgcatt ggttctgcag gactcctcat taagataata atactgg 47 76 19DNA Artificial Sequence Cloning primer 76 cgtctttagc cgggaaacg 19 77 18DNA Artificial Sequence Cloning primer 77 gcagatctcc tggcttgc 18 78 30DNA Artificial Sequence Cloning primer 78 gccgcaagct tcgtctttagccgggaaacg 30 79 26 DNA Artificial Sequence Cloning primer 79 cggtcgacgcagatctcctg gcttgc 26 80 23 DNA Artificial Sequence Cloning primer 80caagccgtca attgtctgat tcg 23 81 22 DNA Artificial Sequence Cloningprimer 81 ggtgaattcc tcctgctagc cc 22 82 34 DNA Artificial SequenceCloning primer 82 gcgccaagct tcaagccgtc aattgtctga ttcg 34 83 28 DNAArtificial Sequence Cloning primer 83 ctgcagggtg aattcctcct gctagccc 2884 42 DNA Artificial Sequence Cloning primer 84 gcttaactcg agcttaataacaagccgtca attgtctgat tc 42 85 34 DNA Artificial Sequence Cloning primer85 gcttaaccgc gggccaagct tgcatgcctg ctcc 34 86 26 DNA ArtificialSequence Cloning primer 86 ggcaaccacg cacgcgcagg gccacc 26 87 25 DNAArtificial Sequence Cloning primer 87 caatggtgat ggtgatgatg accgg 25 8838 DNA Artificial Sequence Cloning primer 88 cgcggtcgac atggcaaccacgcacgcgca gggccacc 38 89 40 DNA Artificial Sequence Cloning primer 89gcgccggtac cttatcaatg gtgatggtga tgatgaccgg 40 90 25 DNA ArtificialSequence Cloning primer 90 ggggcaaccc gggaacggca gcgcc 25 91 30 DNAArtificial Sequence Cloning primer 91 gcagtgagtc atttgtacta caattcctcc30 92 37 DNA Artificial Sequence Cloning primer 92 cgcggtcgac atggggcaacccgggaacgg cagcgcc 37 93 49 DNA Artificial Sequence Cloning primer 93gcgccggatc cttattatag cagtgagtca tttgtactac aattcctcc 49 94 28 DNAArtificial Sequence Cloning primer 94 ggactggtcc ctcacctagg ggacaggg 2895 27 DNA Artificial Sequence Cloning primer 95 ctgagaagac tgggcgcgggcgggagg 27 96 42 DNA Artificial Sequence Cloning primer 96 cgcgggtcgacatgggactg gtccctcacc taggggacag gg 42 97 44 DNA Artificial SequenceCloning primer 97 gcgccggtac cttattactg agaagactgg gcgcgggcgg gagg 44 9816 DNA Artificial Sequence Cloning primer 98 gatagtgtgt gtcccc 16 99 17DNA Artificial Sequence Cloning primer 99 ctgagaagac tgggcgc 17 100 27DNA Artificial Sequence Cloning primer 100 gggagaccat ggatagtgtg tgtcccc27 101 32 DNA Artificial Sequence Cloning primer 101 gcctcatctagattactgag aagactgggc gc 32 102 26 DNA Artificial Sequence Cloningprimer 102 gagcactgaa agcatgatcc gggacg 26 103 28 DNA ArtificialSequence Cloning primer 103 cagggcaatg atcccaaagt agacctgc 28 104 39 DNAArtificial Sequence Cloning primer 104 ccgcggaatt catgagcact gaaagcatgatccgggacg 39 105 43 DNA Artificial Sequence Cloning primer 105ggcgcaagct tatcacaggg caatgatccc aaagtagacc tgc 43 106 110 DNAArtificial Sequence Cloning primer 106 tctgatagcg gtcttacttc ccctcgcagtattactgctc aatagtgact ctgaatgtcc 60 cctgtcccac gatgggtact gcctccatgatggtgtgtgc atgtatattg 110 107 101 DNA Artificial Sequence Cloning primer107 aggtctcggt actgacatcg ctccccgatg tagccaacaa cacagttgca tgcatacttg 60tccaatgctt caatatacat gcacacacca tcatggaggc a 101 108 63 DNA ArtificialSequence Cloning primer 108 ccgcgggtac catgaacttg gggaatcgac tgtttattctgatagcggtc ttacttcccc 60 tcg 63 109 61 DNA Artificial Sequence Cloningprimer 109 gcgccaagct tattagcgca gttcccacca cttcaggtct cggtactgacatcgctcccc 60 g 61 110 22 DNA Artificial Sequence Cloning primer 110tcattcacat tgagcgtcac cg 22 111 24 DNA Artificial Sequence Cloningprimer 111 ttatattgac agcgcacaga gcgg 24 112 101 DNA Artificial SequenceCloning primer 112 gcaagaattc accatgaact tggggaatcg actgtttattctgatagcgg tcttacttcc 60 cctcgcagta ttactgctct cattcacatt gagcgtcacc g101 113 47 DNA Artificial Sequence Cloning primer 113 cgcggttacgtaagcaactg cagttatatt gacagcgcac agagcgg 47 114 24 DNA ArtificialSequence Cloning primer 114 gtcacggccg agacttatag tcgc 24 115 23 DNAArtificial Sequence Cloning primer 115 ggtgtccggg cttttgtcac agg 23 11637 DNA Artificial Sequence Cloning primer 116 cgcggctgca gatgtcacggccgagactta tagtcgc 37 117 38 DNA Artificial Sequence Cloning primer 117cgcggtctag attctggtgt ccgggctttt gtcacagg 38 118 23 DNA ArtificialSequence Cloning primer 118 cagccccaga gcggctttca tgg 23 119 37 DNAArtificial Sequence Cloning primer 119 cgcggtctag atttcagccc cagagcggctttcatgg 37 120 106 DNA Artificial Sequence Cloning primer 120 cgcggctgcagatgaaaata aaaacaggtg cacgcatcct cgcattatcc gcattaacga 60 cgatgatgttttccgcctcg gctctcgcca aaatctctag acgcgg 106 121 106 DNA ArtificialSequence Cloning primer 121 ccgcgtctag agattttggc gagagccgag gcggaaaacatcatcgtcgt taatgcggat 60 aatgcgagga tgcgtgcacc tgtttttatt ttcatctgcagccgcg 106 122 26 DNA Artificial Sequence Cloning primer 122 ggtgcacgcatcctcgcatt atccgc 26 123 26 DNA Artificial Sequence Cloning primer 123cggcatacca gaaagcggac atctgc 26 124 52 DNA Artificial Sequence Cloningprimer 124 cgcggctgca gatgaaaata aaaacaggtg cacgcatcct cgcattatcc gc 52125 42 DNA Artificial Sequence Cloning primer 125 cgcggtctag aacgcacggcataccagaaa gcggacatct gc 42 126 27 DNA Artificial Sequence Cloningprimer 126 cgcgacagcg cgcaataacc gttctcg 27 127 27 DNA ArtificialSequence Cloning primer 127 gctggttcat cagctcgttg aaagtgg 27 128 41 DNAArtificial Sequence Cloning primer 128 gcgccggcgc catacgcgac agcgcgcaataaccgttctc g 41 129 52 DNA Artificial Sequence Cloning primer 129ggcgctctag attattatta cgcctgctgg ttcatcagct cgttgaaagt gg 52 130 29 DNAArtificial Sequence Cloning primer 130 ggtagcacaa tcagattcgc ttatgacgg29 131 25 DNA Artificial Sequence Cloning primer 131 gccgcccatgccacccatgc cgccc 25 132 38 DNA Artificial Sequence Cloning primer 132gcgtctagag gtagcacaat cagattcgct tatgacgg 38 133 49 DNA ArtificialSequence Cloning primer 133 ggcgcaagct tattattaca tcatgccgcc catgccacccatgccgccc 49 134 27 DNA Artificial Sequence Cloning primer 134gcgataaaat tattcacctg actgacg 27 135 26 DNA Artificial Sequence Cloningprimer 135 gcgtcgagga actctttcaa ctgacc 26 136 66 DNA ArtificialSequence Cloning primer 136 cgcggctgca gatgatcgaa gcccgctcta gactcgagagcgataaaatt attcacctga 60 ctgacg 66 137 101 DNA Artificial SequenceCloning primer 137 ccgcgggatc cttattaatc atcatgatct ttataatcgccatcatgatc tttataatcc 60 tcgagcgcca ggttagcgtc gaggaactct ttcaactgac c101 138 65 DNA Artificial Sequence Cloning primer 138 tatgtaaggaggttgtcgac cggctcagtc tagaggtacc cgccctcatc cgaaagggcg 60 tattg 65 13967 DNA Artificial Sequence Cloning primer 139 gatccaatac gccctttcggatgagggcgg gtacctctag actgagccgg tcgacaacct 60 ccttaca 67 140 65 DNAArtificial Sequence Cloning primer 140 tatgtaagga ggttctgcag cggctcagtctagaggtacc cgccctcatc cgaaagggcg 60 tattg 65 141 67 DNA ArtificialSequence Cloning primer 141 gatccaatac gccctttcgg atgagggcgg gtacctctagactgagccgc tgcagaacct 60 ccttaca 67 142 66 DNA Artificial SequenceCloning primer 142 gatcctaagg aggttgtcga ccggctcagt ctagaggtacccgccctcat ccgaaagggc 60 gtattc 66 143 66 DNA Artificial SequenceCloning primer 143 tcgagaatac gccctttcgg atgagggcgg gtacctctagactgagccgg tcgacaacct 60 ccttag 66 144 66 DNA Artificial SequenceCloning primer 144 gatcctaagg aggttctgca gcggctcagt ctagaggtacccgccctcat ccgaaagggc 60 gtattc 66 145 66 DNA Artificial SequenceCloning primer 145 tcgagaatac gccctttcgg atgagggcgg gtacctctagactgagccgc tgcagaacct 60 ccttag 66 146 4740 DNA Artificial SequenceExpression vector 146 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacatgcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccgtcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcagagcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggagaaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatcggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgattaagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaagcttaattaa tctttctgcg 420 aattgagatg acgccactgg ctgggcgtca tcccggtttcccgggtaaac accaccgaaa 480 aatagttact atcttcaaag ccacattcgg tcgaaatatcactgattaac aggcggctat 540 gctggagaag atattgcgca tgacacactc tgacctgtcgcagatattga ttgatggtca 600 ttccagtctg ctggcgaaat tgctgacgca aaacgcgctcactgcacgat gcctcatcac 660 aaaatttatc cagcgcaaag ggacttttca ggctagccgccagccgggta atcagcttat 720 ccagcaacgt ttcgctggat gttggcggca acgaatcactggtgtaacga tggcgattca 780 gcaacatcac caactgcccg aacagcaact cagccatttcgttagcaaac ggcacatgct 840 gactactttc atgctcaagc tgaccgataa cctgccgcgcctgcgccatc cccatgctac 900 ctaagcgcca gtgtggttgc cctgcgctgg cgttaaatcccggaatcgcc ccctgccagt 960 caagattcag cttcagacgc tccgggcaat aaataatattctgcaaaacc agatcgttaa 1020 cggaagcgta ggagtgttta tcgtcagcat gaatgtaaaagagatcgcca cgggtaatgc 1080 gataagggcg atcgttgagt acatgcaggc cattaccgcgccagacaatc accagctcac 1140 aaaaatcatg tgtatgttca gcaaagacat cttgcggataacggtcagcc acagcgactg 1200 cctgctggtc gctggcaaaa aaatcatctt tgagaagttttaactgatgc gccaccgtgg 1260 ctacctcggc cagagaacga agttgattat tcgcaatatggcgtacaaat acgttgagaa 1320 gattcgcgtt attgcagaaa gccatcccgt ccctggcgaatatcacgcgg tgaccagtta 1380 aactctcggc gaaaaagcgt cgaaaagtgg ttactgtcgctgaatccaca gcgataggcg 1440 atgtcagtaa cgctggcctc gctgtggcgt agcagatgtcgggctttcat cagtcgcagg 1500 cggttcaggt atcgctgagg cgtcagtccc gtttgctgcttaagctgccg atgtagcgta 1560 cgcagtgaaa gagaaaattg atccgccacg gcatcccaattcacctcatc ggcaaaatgg 1620 tcctccagcc aggccagaag caagttgaga cgtgatgcgctgttttccag gttctcctgc 1680 aaactgcttt tacgcagcaa gagcagtaat tgcataaacaagatctcgcg actggcggtc 1740 gagggtaaat cattttcccc ttcctgctgt tccatctgtgcaaccagctg tcgcacctgc 1800 tgcaatacgc tgtggttaac gcgccagtga gacggatactgcccatccag ctcttgtggc 1860 agcaactgat tcagcccggc gagaaactga aatcgatccggcgagcgata cagcacattg 1920 gtcagacaca gattatcggt atgttcatac agatgccgatcatgatcgcg tacgaaacag 1980 accgtgccac cggtgatggt atagggctgc ccattaaacacatgaatacc cgtgccatgt 2040 tcgacaatca caatttcatg aaaatcatga tgatgttcaggaaaatccgc ctgcgggagc 2100 cggggttcta tcgccacgga cgcgttacca gacggaaaaaaatccacact atgtaatacg 2160 gtcatactgg cctcctgatg tcgtcaacac ggcgaaatagtaatcacgag gtcaggttct 2220 taccttaaat tttcgacgga aaaccacgta aaaaacgtcgatttttcaag atacagcgtg 2280 aattttcagg aaatgcggtg agcatcacat caccacaattcagcaaattg tgaacatcat 2340 cacgttcatc tttccctggt tgccaatggc ccattttcctgtcagtaacg agaaggtcgc 2400 gaattcaggc gctttttaga ctggtcgtaa tgaaattcaggaggttgtcg actctagagg 2460 atccccgcgc cctcatccga aagggcgtat tggtaccgagctcgaattcg taatcatggt 2520 catagctgtt tcctgtgtga aattgttatc cgctcacaattccacacaac atacgagccg 2580 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgagctaactcaca ttaattgcgt 2640 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtgccagctgcat taatgaatcg 2700 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctcttccgcttcc tcgctcactg 2760 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatcagctcactca aaggcggtaa 2820 tacggttatc cacagaatca ggggataacg caggaaagaacatgtgagca aaaggccagc 2880 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtttttccatagg ctccgccccc 2940 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtggcgaaacccg acaggactat 3000 aaagatacca ggcgtttccc cctggaagct ccctcgtgcgctctcctgtt ccgaccctgc 3060 cgcttaccgg atacctgtcc gcctttctcc cttcgggaagcgtggcgctt tctcatagct 3120 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctccaagctgggc tgtgtgcacg 3180 aaccccccgt tcagcccgac cgctgcgcct tatccggtaactatcgtctt gagtccaacc 3240 cggtaagaca cgacttatcg ccactggcag cagccactggtaacaggatt agcagagcga 3300 ggtatgtagg cggtgctaca gagttcttga agtggtggcctaactacggc tacactagaa 3360 ggacagtatt tggtatctgc gctctgctga agccagttaccttcggaaaa agagttggta 3420 gctcttgatc cggcaaacaa accaccgctg gtagcggtggtttttttgtt tgcaagcagc 3480 agattacgcg cagaaaaaaa ggatctcaag aagatcctttgatcttttct acggggtctg 3540 acgctcagtg gaacgaaaac tcacgttaag ggattttggtcatgagatta tcaaaaagga 3600 tcttcaccta gatcctttta aattaaaaat gaagttttaaatcaatctaa agtatatatg 3660 agtaaacttg gtctgacagt taccaatgct taatcagtgaggcacctatc tcagcgatct 3720 gtctatttcg ttcatccata gttgcctgac tccccgtcgtgtagataact acgatacggg 3780 agggcttacc atctggcccc agtgctgcaa tgataccgcgagacccacgc tcaccggctc 3840 cagatttatc agcaataaac cagccagccg gaagggccgagcgcagaagt ggtcctgcaa 3900 ctttatccgc ctccatccag tctattaatt gttgccgggaagctagagta agtagttcgc 3960 cagttaatag tttgcgcaac gttgttgcca ttgctacaggcatcgtggtg tcacgctcgt 4020 cgtttggtat ggcttcattc agctccggtt cccaacgatcaaggcgagtt acatgatccc 4080 ccatgttgtg caaaaaagcg gttagctcct tcggtcctccgatcgttgtc agaagtaagt 4140 tggccgcagt gttatcactc atggttatgg cagcactgcataattctctt actgtcatgc 4200 catccgtaag atgcttttct gtgactggtg agtactcaaccaagtcattc tgagaatagt 4260 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacgggataatacc gcgccacata 4320 gcagaacttt aaaagtgctc atcattggaa aacgttcttcggggcgaaaa ctctcaagga 4380 tcttaccgct gttgagatcc agttcgatgt aacccactcgtgcacccaac tgatcttcag 4440 catcttttac tttcaccagc gtttctgggt gagcaaaaacaggaaggcaa aatgccgcaa 4500 aaaagggaat aagggcgaca cggaaatgtt gaatactcatactcttcctt tttcaatatt 4560 attgaagcat ttatcagggt tattgtctca tgagcggatacatatttgaa tgtatttaga 4620 aaaataaaca aataggggtt ccgcgcacat ttccccgaaaagtgccacct gacgtctaag 4680 aaaccattat tatcatgaca ttaacctata aaaataggcgtatcacgagg ccctttcgtc 4740 147 4746 DNA Artificial Sequence Expressionvector 147 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccggagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcgtcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgtactgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgcatcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcctcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggtaacgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttaattaatctttctgcg 420 aattgagatg acgccactgg ctgggcgtca tcccggtttc ccgggtaaacaccaccgaaa 480 aatagttact atcttcaaag ccacattcgg tcgaaatatc actgattaacaggcggctat 540 gctggagaag atattgcgca tgacacactc tgacctgtcg cagatattgattgatggtca 600 ttccagtctg ctggcgaaat tgctgacgca aaacgcgctc actgcacgatgcctcatcac 660 aaaatttatc cagcgcaaag ggacttttca ggctagccgc cagccgggtaatcagcttat 720 ccagcaacgt ttcgctggat gttggcggca acgaatcact ggtgtaacgatggcgattca 780 gcaacatcac caactgcccg aacagcaact cagccatttc gttagcaaacggcacatgct 840 gactactttc atgctcaagc tgaccgataa cctgccgcgc ctgcgccatccccatgctac 900 ctaagcgcca gtgtggttgc cctgcgctgg cgttaaatcc cggaatcgccccctgccagt 960 caagattcag cttcagacgc tccgggcaat aaataatatt ctgcaaaaccagatcgttaa 1020 cggaagcgta ggagtgttta tcgtcagcat gaatgtaaaa gagatcgccacgggtaatgc 1080 gataagggcg atcgttgagt acatgcaggc cattaccgcg ccagacaatcaccagctcac 1140 aaaaatcatg tgtatgttca gcaaagacat cttgcggata acggtcagccacagcgactg 1200 cctgctggtc gctggcaaaa aaatcatctt tgagaagttt taactgatgcgccaccgtgg 1260 ctacctcggc cagagaacga agttgattat tcgcaatatg gcgtacaaatacgttgagaa 1320 gattcgcgtt attgcagaaa gccatcccgt ccctggcgaa tatcacgcggtgaccagtta 1380 aactctcggc gaaaaagcgt cgaaaagtgg ttactgtcgc tgaatccacagcgataggcg 1440 atgtcagtaa cgctggcctc gctgtggcgt agcagatgtc gggctttcatcagtcgcagg 1500 cggttcaggt atcgctgagg cgtcagtccc gtttgctgct taagctgccgatgtagcgta 1560 cgcagtgaaa gagaaaattg atccgccacg gcatcccaat tcacctcatcggcaaaatgg 1620 tcctccagcc aggccagaag caagttgaga cgtgatgcgc tgttttccaggttctcctgc 1680 aaactgcttt tacgcagcaa gagcagtaat tgcataaaca agatctcgcgactggcggtc 1740 gagggtaaat cattttcccc ttcctgctgt tccatctgtg caaccagctgtcgcacctgc 1800 tgcaatacgc tgtggttaac gcgccagtga gacggatact gcccatccagctcttgtggc 1860 agcaactgat tcagcccggc gagaaactga aatcgatccg gcgagcgatacagcacattg 1920 gtcagacaca gattatcggt atgttcatac agatgccgat catgatcgcgtacgaaacag 1980 accgtgccac cggtgatggt atagggctgc ccattaaaca catgaatacccgtgccatgt 2040 tcgacaatca caatttcatg aaaatcatga tgatgttcag gaaaatccgcctgcgggagc 2100 cggggttcta tcgccacgga cgcgttacca gacggaaaaa aatccacactatgtaatacg 2160 gtcatactgg cctcctgatg tcgtcaacac ggcgaaatag taatcacgaggtcaggttct 2220 taccttaaat tttcgacgga aaaccacgta aaaaacgtcg atttttcaagatacagcgtg 2280 aattttcagg aaatgcggtg agcatcacat caccacaatt cagcaaattgtgaacatcat 2340 cacgttcatc tttccctggt tgccaatggc ccattttcct gtcagtaacgagaaggtcgc 2400 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag gaggttctgcaggtcgactc 2460 tagaggatcc ccgcgccctc atccgaaagg gcgtattggt accgagctcgaattcgtaat 2520 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattccacacaacatac 2580 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaactcacattaa 2640 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccagctgcattaat 2700 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttccgcttcctcgc 2760 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagctcactcaaagg 2820 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatgtgagcaaaag 2880 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttccataggctcc 2940 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcgaaacccgacag 3000 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctctcctgttccga 3060 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtggcgctttctc 3120 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaagctgggctgtg 3180 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactatcgtcttgagt 3240 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaacaggattagca 3300 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaactacggctaca 3360 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttcggaaaaagag 3420 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttttttgtttgca 3480 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatcttttctacgg 3540 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatgagattatcaa 3600 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatcaatctaaagta 3660 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggcacctatctcag 3720 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtagataactacga 3780 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagacccacgctcac 3840 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgcagaagtggtc 3900 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagctagagtaagta 3960 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatcgtggtgtcac 4020 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaaggcgagttacat 4080 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatcgttgtcagaa 4140 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataattctcttactg 4200 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaagtcattctgag 4260 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggataataccgcgc 4320 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcggggcgaaaactct 4380 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgcacccaactgat 4440 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacaggaaggcaaaatg 4500 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactcttcctttttc 4560 aatattattg aagcatttat cagggttatt gtctcatgag cggatacatatttgaatgta 4620 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtgccacctgacg 4680 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatcacgaggccct 4740 ttcgtc 4746 148 3946 DNA Artificial Sequence Expressionvector 148 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccggagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcgtcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgtactgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgcatcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcctcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggtaacgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttcaagccgtcaattgtc 420 tgattcgtta ccaattatga caacttgacg gctacatcat tcactttttcttcacaaccg 480 gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcgagaaatagagt 540 tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggtggtgctcaaa 600 agcagcttcg cctggctgat acgttggtcc tcgcgccagc ttaagacgctaatccctaac 660 tgctggcgga aaagatgtga cagacgcgac ggcgacaagc aaacatgctgtgcgacgctg 720 gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagcctcgcgtacc 780 cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccgcagtaacaat 840 tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttgcccggcgtta 900 atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgggcgaaagaac 960 cccgtattgg caaatattga cggccagtta agccattcat gccagtaggcgcgcggacga 1020 aagtaaaccc actggtgata ccattcgcga gcctccggat gacgaccgtagtgatgaatc 1080 tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcgtccctgattt 1140 ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcattcccagcggt 1200 cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacccgccaccaga 1260 tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagccatacttttc 1320 atactcccgc cattcagaga agaaaccaat tgtccatatt gcatcagacattgccgtcac 1380 tgcgtctttt actggctctt ctcgctaacc aaaccggtaa ccccgcttattaaaagcatt 1440 ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta acaaaagtgtctataatcac 1500 ggcagaaaag tccacattga ttatttgcac ggcgtcacac tttgctatgccatagcattt 1560 ttatccataa gattagcgga tcctacctga cgctttttat cgcaactctctactgtttct 1620 ccatacccgt ttttttgggc tagcaggagg ccgtcgactc tagaggatccccgcgccctc 1680 atccgaaagg gcgtattggt accgagctcg aattcgtaat catggtcatagctgtttcct 1740 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaagcataaagtgt 1800 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcgctcactgccc 1860 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggccaacgcgcgggg 1920 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactcgctgcgctcg 1980 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacggttatccaca 2040 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaaggccaggaac 2100 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctgacgagcatcac 2160 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaagataccaggcg 2220 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgcttaccggatac 2280 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacgctgtaggtat 2340 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaaccccccgttcag 2400 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggtaagacacgac 2460 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggtatgtaggcggt 2520 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggacagtatttggt 2580 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctcttgatccggc 2640 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagattacgcgcaga 2700 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgctcagtggaac 2760 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatcttcacctagatc 2820 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagtaaacttggtct 2880 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtctatttcgttca 2940 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggagggcttaccatct 3000 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccagatttatcagca 3060 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaactttatccgcctcc 3120 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagttaatagtttg 3180 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtttggtatggct 3240 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccatgttgtgcaaa 3300 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggccgcagtgtta 3360 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatccgtaagatgc 3420 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtatgcggcgaccg 3480 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcagaactttaaaa 3540 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatcttaccgctgttg 3600 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatcttttactttc 3660 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaagggaataagg 3720 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattgaagcatttat 3780 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaataaacaaata 3840 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaaccattattatc 3900 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 3946149 3952 DNA Artificial Sequence Expression vector 149 tcgcgcgtttcggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtctgtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtgtcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcggtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccattcaggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagctggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtcacgacgttgt aaaacgacgg ccagtgccaa gcttcaagcc gtcaattgtc 420 tgattcgttaccaattatga caacttgacg gctacatcat tcactttttc ttcacaaccg 480 gcacggaactcgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt 540 tgatcgtcaaaaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa 600 agcagcttcgcctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac 660 tgctggcggaaaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg 720 gcgatatcaaaattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc 780 cgattatccatcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat 840 tgctcaagcagatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta 900 atgatttgcccaaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac 960 cccgtattggcaaatattga cggccagtta agccattcat gccagtaggc gcgcggacga 1020 aagtaaacccactggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc 1080 tctcctggcgggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt 1140 ttcaccaccccctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt 1200 cggtcgataaaaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga 1260 tgggcattaaacgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc 1320 atactcccgccattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac 1380 tgcgtcttttactggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt 1440 ctgtaacaaagcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac 1500 ggcagaaaagtccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt 1560 ttatccataagattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct 1620 ccatacccgtttttttgggc tagcaggagg ccctgcaggt cgactctaga ggatccccgc 1680 gccctcatccgaaagggcgt attggtaccg agctcgaatt cgtaatcatg gtcatagctg 1740 tttcctgtgtgaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 1800 aagtgtaaagcctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 1860 ctgcccgctttccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 1920 gcggggagaggcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 1980 cgctcggtcgttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 2040 tccacagaatcaggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 2100 aggaaccgtaaaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 2160 catcacaaaaatcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 2220 caggcgtttccccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 2280 ggatacctgtccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 2340 aggtatctcagttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 2400 gttcagcccgaccgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 2460 cacgacttatcgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 2520 ggcggtgctacagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 2580 tttggtatctgcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 2640 tccggcaaacaaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 2700 cgcagaaaaaaaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 2760 tggaacgaaaactcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 2820 tagatccttttaaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 2880 tggtctgacagttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 2940 cgttcatccatagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 3000 ccatctggccccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 3060 tcagcaataaaccagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 3120 gcctccatccagtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 3180 agtttgcgcaacgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 3240 atggcttcattcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 3300 tgcaaaaaagcggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 3360 gtgttatcactcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 3420 agatgcttttctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 3480 cgaccgagttgctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 3540 ttaaaagtgctcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 3600 ctgttgagatccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 3660 actttcaccagcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 3720 ataagggcgacacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 3780 atttatcagggttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 3840 caaataggggttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 3900 attatcatgacattaaccta taaaaatagg cgtatcacga ggccctttcg tc 3952 150 3886 DNAArtificial Sequence Expression vector 150 tcgcgcgttt cggtgatgacggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggatgccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctggcttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaataccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgcaactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaagggggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgtaaaacgacgg ccagtgccaa gcttttagcc gggaaacgtc 420 tggcggcgct gttggctaagtttgcggtat tgttgcggcg acatgccgac atatttgccg 480 aacgtgctgt aaaaacgactacttgaacga aagcctgccg tcagggcaat atcgagaata 540 cttttatcgg tatcgctcagtaacgcgcga acgtggttga tgcgcatcgc ggtaatgtac 600 tgtttcatcg tcaattgcatgacccgctgg aatatcccca ttgcatagtt ggcgttaagt 660 ttgacgtgct cagccacatcgttgatggtc agcgcctgat catagttttc ggcaataaag 720 cccagcatct ggctaacataaaattgcgca tggcgcgaga cgctgttttt gtgtgtgcgc 780 gaggttttat tgaccagaatcggttcccag ccagagaggc taaatcgctt gagcatcagg 840 ccaatttcat caatggcgagctggcgaatt tgctcgttcg gactgtttaa ttcctgctgc 900 cagcggcgca cttcaaacgggctaagttgc tgtgtggcca gtgatttgat caccatgccg 960 tgagtgacgt ggttaatcaggtctttatcc agcggccagg agagaaacag atgcatcggc 1020 agattaaaaa tcgccatgctctgacaggtt ccggtatctg ttagttggtg cggtgtacag 1080 gcccagaaca gcgtgatatgaccctgattg atattcactt tttcattgtt gatcaggtat 1140 tccacatcgc catcgaaaggcacattcact tcgacctgac catgccagtg gctggtgggc 1200 atgatatgcg gtgcgcgaaactcaatctcc atccgctggt attccgaata cagcgacagc 1260 gggctgcggg tctgtttttcgtcgctgctg cacataaacg tatctgtatt catggatggc 1320 tctctttcct ggaatatcagaattatggca ggagtgaggg aggatgactg cgagtgggag 1380 cacggttttc accctcttcccagaggggcg aggggactct ccgagtatca tgaggccgaa 1440 aactctgctt ttcaggtaatttattcccat aaactcagat ttactgctgc ttcacgcagg 1500 atctgagttt atgggaatgctcaacctgga agccggaggt tttctgcaga ttcgcctgcc 1560 atgatgaagt tattcaagcaagccaggagg tcgtcgactc tagaggatcc ccgcgccctc 1620 atccgaaagg gcgtattggtaccgagctcg aattcgtaat catggtcata gctgtttcct 1680 gtgtgaaatt gttatccgctcacaattcca cacaacatac gagccggaag cataaagtgt 1740 aaagcctggg gtgcctaatgagtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 1800 gctttccagt cgggaaacctgtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 1860 agaggcggtt tgcgtattgggcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 1920 gtcgttcggc tgcggcgagcggtatcagct cactcaaagg cggtaatacg gttatccaca 1980 gaatcagggg ataacgcaggaaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 2040 cgtaaaaagg ccgcgttgctggcgtttttc cataggctcc gcccccctga cgagcatcac 2100 aaaaatcgac gctcaagtcagaggtggcga aacccgacag gactataaag ataccaggcg 2160 tttccccctg gaagctccctcgtgcgctct cctgttccga ccctgccgct taccggatac 2220 ctgtccgcct ttctcccttcgggaagcgtg gcgctttctc atagctcacg ctgtaggtat 2280 ctcagttcgg tgtaggtcgttcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 2340 cccgaccgct gcgccttatccggtaactat cgtcttgagt ccaacccggt aagacacgac 2400 ttatcgccac tggcagcagccactggtaac aggattagca gagcgaggta tgtaggcggt 2460 gctacagagt tcttgaagtggtggcctaac tacggctaca ctagaaggac agtatttggt 2520 atctgcgctc tgctgaagccagttaccttc ggaaaaagag ttggtagctc ttgatccggc 2580 aaacaaacca ccgctggtagcggtggtttt tttgtttgca agcagcagat tacgcgcaga 2640 aaaaaaggat ctcaagaagatcctttgatc ttttctacgg ggtctgacgc tcagtggaac 2700 gaaaactcac gttaagggattttggtcatg agattatcaa aaaggatctt cacctagatc 2760 cttttaaatt aaaaatgaagttttaaatca atctaaagta tatatgagta aacttggtct 2820 gacagttacc aatgcttaatcagtgaggca cctatctcag cgatctgtct atttcgttca 2880 tccatagttg cctgactccccgtcgtgtag ataactacga tacgggaggg cttaccatct 2940 ggccccagtg ctgcaatgataccgcgagac ccacgctcac cggctccaga tttatcagca 3000 ataaaccagc cagccggaagggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 3060 atccagtcta ttaattgttgccgggaagct agagtaagta gttcgccagt taatagtttg 3120 cgcaacgttg ttgccattgctacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 3180 tcattcagct ccggttcccaacgatcaagg cgagttacat gatcccccat gttgtgcaaa 3240 aaagcggtta gctccttcggtcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 3300 tcactcatgg ttatggcagcactgcataat tctcttactg tcatgccatc cgtaagatgc 3360 ttttctgtga ctggtgagtactcaaccaag tcattctgag aatagtgtat gcggcgaccg 3420 agttgctctt gcccggcgtcaatacgggat aataccgcgc cacatagcag aactttaaaa 3480 gtgctcatca ttggaaaacgttcttcgggg cgaaaactct caaggatctt accgctgttg 3540 agatccagtt cgatgtaacccactcgtgca cccaactgat cttcagcatc ttttactttc 3600 accagcgttt ctgggtgagcaaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 3660 gcgacacgga aatgttgaatactcatactc ttcctttttc aatattattg aagcatttat 3720 cagggttatt gtctcatgagcggatacata tttgaatgta tttagaaaaa taaacaaata 3780 ggggttccgc gcacatttccccgaaaagtg ccacctgacg tctaagaaac cattattatc 3840 atgacattaa cctataaaaataggcgtatc acgaggccct ttcgtc 3886 151 28 DNA Artificial Sequence Cloningprimer 151 gcagaacctc ctgaatttca ttacgacc 28 152 86 DNA ArtificialSequence Cloning primer 152 ccgcgggtac caatacgccc tttcggatga gggcgcggggatcctctaga gtcgacgtcg 60 acaacctcct gaatttcatt acgacc 86 153 86 DNAArtificial Sequence Cloning primer 153 ccgcgggtac caatacgccc tttcggatgagggcgcgggg atcctctaga gtcgacctgc 60 agaacctcct gaatttcatt acgacc 86 15438 DNA Artificial Sequence Cloning primer 154 ctgcagggcc tcctgctagcccaaaaaaac gggtatgg 38 155 94 DNA Artificial Sequence Cloning primer 155ccgcgggtac caatacgccc tttcggatga gggcgcgggg atcctctaga gtcgacgtcg 60acggcctcct gctagcccaa aaaaacgggt atgg 94 156 94 DNA Artificial SequenceCloning primer 156 ccgcgggtac caatacgccc tttcggatga gggcgcggggatcctctaga gtcgacctgc 60 agggcctcct gctagcccaa aaaaacgggt atgg 94 157 32DNA Artificial Sequence Cloning primer 157 cctcctggct tgcttgaataacttcatcat gg 32 158 91 DNA Artificial Sequence Cloning primer 158ccgcgggtac caatacgccc tttcggatga gggcgcgggg atcctctaga gtcgaccccc 60tcctggcttg cttgaataac ttcatcatgg c 91 159 2319 DNA Artificial SequenceFusion protein 159 gtcgacatga aaataaaaac aggtgcacgc atcctcgcattatccgcatt aacgacgatg 60 atgttttccg cctcggctct cgccaaaatc gaagaaggtaaactggtaat ctggattaac 120 ggcgataaag gctataacgg tctcgctgaa gtcggtaagaaattcgagaa agataccgga 180 attaaagtca ccgttgagca tccggataaa ctggaagagaaattcccaca ggttgcggca 240 actggcgatg gccctgacat tatcttctgg gcacacgaccgctttggtgg ctacgctcaa 300 tctggcctgt tggctgaaat caccccggac aaagcgttccaggacaagct gtatccgttt 360 acctgggatg ccgtacgtta caacggcaag ctgattgcttacccgatcgc tgttgaagcg 420 ttatcgctga tttataacaa agatctgctg ccgaacccgccaaaaacctg ggaagagatc 480 ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcgcgctgatgtt caacctgcaa 540 gaaccgtact tcacctggcc gctgattgct gctgacgggggttatgcgtt caagtatgaa 600 aacggcaagt acgacattaa agacgtgggc gtggataacgctggcgcgaa agcgggtctg 660 accttcctgg ttgacctgat taaaaacaaa cacatgaatgcagacaccga ttactccatc 720 gcagaagctg cctttaataa aggcgaaaca gcgatgaccatcaacggccc gtgggcatgg 780 tccaacatcg acaccagcaa agtgaattat ggtgtaacggtactgccgac cttcaagggt 840 caaccatcca aaccgttcgt tggcgtgctg agcgcaggtattaacgccgc cagtccgaac 900 aaagagctgg cgaaagagtt cctcgaaaac tatctgctgactgatgaagg tctggaagcg 960 gttaataaag acaaaccgct gggtgccgta gcgctgaagtcttacgagga agagttggcg 1020 aaagatccac gtattgccgc caccatggaa aacgcccagaaaggtgaaat catgccgaac 1080 atcccgcaga tgtccgcttt ctggtatgcc gtgctgatcgaagcccgcac ctcggaatcc 1140 gacacggcag ggcccaacag cgacctggac gtgaacactgacatttattc caaggtgctg 1200 gtgactgcta tatacctggc actcttcgtg gtgggcactgtgggcaactc cgtgacagcc 1260 ttcactctag cgcggaagaa gtcactgcag agcctgcagagcactgtgca ttaccacctg 1320 ggcagcctgg cactgtcgga cctgcttatc cttctgctggccatgcccgt ggagctatac 1380 aacttcatct gggtacacca tccctgggcc tttggggacgctggctgccg tggctactat 1440 ttcctgcgtg atgcctgcac ctatgccaca gccctcaatgtagccagcct gagtgtggag 1500 cgctacttgg ccatctgcca tcccttcaag gccaagaccctcatgtcccg cagccgcacc 1560 aagaaattca tcagtgccat atggctagct tcggcgctgctggctatacc catgcttttc 1620 accatgggcc tgcagaaccg cagtggtgac ggcacgcaccctggcggcct ggtgtgcaca 1680 cccattgtgg acacagccac tgtcaaggtc gtcatccaggttaacacctt catgtccttc 1740 ctgtttccca tgttggtcat ctccatccta aacaccgtgattgccaacaa actgacagtc 1800 atggtgcacc aggccgccga gcagggccga gtgtgcaccgtgggcacaca caacggttta 1860 gagcacagca cgttcaacat gaccatcgag ccgggtcgtgtccaggccct gcgccacgga 1920 gtcctcgtct tacgtgctgt ggtcattgcc tttgtggtctgctggctgcc ctaccacgtg 1980 cgacgcctga tgttctgcta tatctcggat gaacagtggactacgttcct cttcgatttc 2040 taccactatt tctacatgct aaccaacgct ctcttctacgtcagctccgc catcaatccc 2100 atcctctaca acctggtctc cgccaacttc cgccaggtctttctgtccac gctggcctgc 2160 ctttgtcctg ggtggcgcca ccgccgaaag aagaggccaacgttctccag gaagcccaac 2220 agcatgtcca gcaaccatgc cttttccacc agcgccacccgggagaccct gtacgcggcc 2280 gcagattata aagatgacga tgacaaataa taaggtacc2319 160 1293 DNA Artificial Sequence Fusion protein 160 gtcgacatgaaaataaaaac aggtgcacgc atcctcgcat tatccgcatt aacgacgatg 60 atgttttccgcctcggctct cgccaaaatc atcgaagccc gcacctcgga atccgacacg 120 gcagggcccaacagcgacct ggacgtgaac actgacattt attccaaggt gctggtgact 180 gctatatacctggcactctt cgtggtgggc actgtgggca actccgtgac agccttcact 240 ctagcgcggaagaagtcact gcagagcctg cagagcactg tgcattacca cctgggcagc 300 ctggcactgtcggacctgct tatccttctg ctggccatgc ccgtggagct atacaacttc 360 atctgggtacaccatccctg ggcctttggg gacgctggct gccgtggcta ctatttcctg 420 cgtgatgcctgcacctatgc cacagccctc aatgtagcca gcctgagtgt ggagcgctac 480 ttggccatctgccatccctt caaggccaag accctcatgt cccgcagccg caccaagaaa 540 ttcatcagtgccatatggct agcttcggcg ctgctggcta tacccatgct tttcaccatg 600 ggcctgcagaaccgcagtgg tgacggcacg caccctggcg gcctggtgtg cacacccatt 660 gtggacacagccactgtcaa ggtcgtcatc caggttaaca ccttcatgtc cttcctgttt 720 cccatgttggtcatctccat cctaaacacc gtgattgcca acaaactgac agtcatggtg 780 caccaggccgccgagcaggg ccgagtgtgc accgtgggca cacacaacgg tttagagcac 840 agcacgttcaacatgaccat cgagccgggt cgtgtccagg ccctgcgcca cggagtcctc 900 gtcttacgtgctgtggtcat tgcctttgtg gtctgctggc tgccctacca cgtgcgacgc 960 ctgatgttctgctatatctc ggatgaacag tggactacgt tcctcttcga tttctaccac 1020 tatttctacatgctaaccaa cgctctcttc tacgtcagct ccgccatcaa tcccatcctc 1080 tacaacctggtctccgccaa cttccgccag gtctttctgt ccacgctggc ctgcctttgt 1140 cctgggtggcgccaccgccg aaagaagagg ccaacgttct ccaggaagcc caacagcatg 1200 tccagcaaccatgccttttc caccagcgcc acccgggaga ccctgtacgc ggccgcagat 1260 tataaagatgacgatgacaa ataataaggt acc 1293 161 2652 DNA Artificial Sequence Fusionprotein 161 gtcgacatga aaataaaaac aggtgcacgc atcctcgcat tatccgcattaacgacgatg 60 atgttttccg cctcggctct cgccaaaatc gaagaaggta aactggtaatctggattaac 120 ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaaagataccgga 180 attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccacaggttgcggca 240 actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtggctacgctcaa 300 tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagctgtatccgttt 360 acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgctgttgaagcg 420 ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctgggaagagatc 480 ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgttcaacctgcaa 540 gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgttcaagtatgaa 600 aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaaagcgggtctg 660 accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccgattactccatc 720 gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggcccgtgggcatgg 780 tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgaccttcaagggt 840 caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgccagtccgaac 900 aaagagctgg cgaaagagtt cctcgaaaac tatctgctga ctgatgaaggtctggaagcg 960 gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgaggaagagttggcg 1020 aaagatccac gtattgccgc caccatggaa aacgcccaga aaggtgaaatcatgccgaac 1080 atcccgcaga tgtccgcttt ctggtatgcc gtgctgatcg aagcccgcacctcggaatcc 1140 gacacggcag ggcccaacag cgacctggac gtgaacactg acatttattccaaggtgctg 1200 gtgactgcta tatacctggc actcttcgtg gtgggcactg tgggcaactccgtgacagcc 1260 ttcactctag cgcggaagaa gtcactgcag agcctgcaga gcactgtgcattaccacctg 1320 ggcagcctgg cactgtcgga cctgcttatc cttctgctgg ccatgcccgtggagctatac 1380 aacttcatct gggtacacca tccctgggcc tttggggacg ctggctgccgtggctactat 1440 ttcctgcgtg atgcctgcac ctatgccaca gccctcaatg tagccagcctgagtgtggag 1500 cgctacttgg ccatctgcca tcccttcaag gccaagaccc tcatgtcccgcagccgcacc 1560 aagaaattca tcagtgccat atggctagct tcggcgctgc tggctatacccatgcttttc 1620 accatgggcc tgcagaaccg cagtggtgac ggcacgcacc ctggcggcctggtgtgcaca 1680 cccattgtgg acacagccac tgtcaaggtc gtcatccagg ttaacaccttcatgtccttc 1740 ctgtttccca tgttggtcat ctccatccta aacaccgtga ttgccaacaaactgacagtc 1800 atggtgcacc aggccgccga gcagggccga gtgtgcaccg tgggcacacacaacggttta 1860 gagcacagca cgttcaacat gaccatcgag ccgggtcgtg tccaggccctgcgccacgga 1920 gtcctcgtct tacgtgctgt ggtcattgcc tttgtggtct gctggctgccctaccacgtg 1980 cgacgcctga tgttctgcta tatctcggat gaacagtgga ctacgttcctcttcgatttc 2040 taccactatt tctacatgct aaccaacgct ctcttctacg tcagctccgccatcaatccc 2100 atcctctaca acctggtctc cgccaacttc cgccaggtct ttctgtccacgctggcctgc 2160 ctttgtcctg ggtggcgcca ccgccgaaag aagaggccaa cgttctccaggaagcccaac 2220 agcatgtcca gcaaccatgc cttttccacc agcgccaccc gggagaccctgtacgcggcc 2280 gcaagcgata aaattattca cctgactgac gacagttttg acacggatgtactcaaagcg 2340 gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaaaatgatcgcc 2400 ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgcaaaactgaac 2460 atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatcccgactctgctg 2520 ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaaaggtcagttg 2580 aaagagttcc tcgacgctaa cctggcggcg gccgcagatt ataaagatgacgatgacaaa 2640 taataaggta cc 2652 162 1626 DNA Artificial SequenceFusion protein 162 gtcgacatga aaataaaaac aggtgcacgc atcctcgcattatccgcatt aacgacgatg 60 atgttttccg cctcggctct cgccaaaatc atcgaagcccgcacctcgga atccgacacg 120 gcagggccca acagcgacct ggacgtgaac actgacatttattccaaggt gctggtgact 180 gctatatacc tggcactctt cgtggtgggc actgtgggcaactccgtgac agccttcact 240 ctagcgcgga agaagtcact gcagagcctg cagagcactgtgcattacca cctgggcagc 300 ctggcactgt cggacctgct tatccttctg ctggccatgcccgtggagct atacaacttc 360 atctgggtac accatccctg ggcctttggg gacgctggctgccgtggcta ctatttcctg 420 cgtgatgcct gcacctatgc cacagccctc aatgtagccagcctgagtgt ggagcgctac 480 ttggccatct gccatccctt caaggccaag accctcatgtcccgcagccg caccaagaaa 540 ttcatcagtg ccatatggct agcttcggcg ctgctggctatacccatgct tttcaccatg 600 ggcctgcaga accgcagtgg tgacggcacg caccctggcggcctggtgtg cacacccatt 660 gtggacacag ccactgtcaa ggtcgtcatc caggttaacaccttcatgtc cttcctgttt 720 cccatgttgg tcatctccat cctaaacacc gtgattgccaacaaactgac agtcatggtg 780 caccaggccg ccgagcaggg ccgagtgtgc accgtgggcacacacaacgg tttagagcac 840 agcacgttca acatgaccat cgagccgggt cgtgtccaggccctgcgcca cggagtcctc 900 gtcttacgtg ctgtggtcat tgcctttgtg gtctgctggctgccctacca cgtgcgacgc 960 ctgatgttct gctatatctc ggatgaacag tggactacgttcctcttcga tttctaccac 1020 tatttctaca tgctaaccaa cgctctcttc tacgtcagctccgccatcaa tcccatcctc 1080 tacaacctgg tctccgccaa cttccgccag gtctttctgtccacgctggc ctgcctttgt 1140 cctgggtggc gccaccgccg aaagaagagg ccaacgttctccaggaagcc caacagcatg 1200 tccagcaacc atgccttttc caccagcgcc acccgggagaccctgtacgc ggccgcaagc 1260 gataaaatta ttcacctgac tgacgacagt tttgacacggatgtactcaa agcggacggg 1320 gcgatcctcg tcgatttctg ggcagagtgg tgcggtccgtgcaaaatgat cgccccgatt 1380 ctggatgaaa tcgctgacga atatcagggc aaactgaccgttgcaaaact gaacatcgat 1440 caaaaccctg gcactgcgcc gaaatatggc atccgtggtatcccgactct gctgctgttc 1500 aaaaacggtg aagtggcggc aaccaaagtg ggtgcactgtctaaaggtca gttgaaagag 1560 ttcctcgacg ctaacctggc agcggccgca gattataaagatgacgatga caaataataa 1620 ggtacc 1626 163 26 DNA Artificial SequenceCloning primer 163 ggtgcacgca tcctcgcatt atccgc 26 164 31 DNA ArtificialSequence Cloning primer 164 cgcacggcat accagaaagc ggacatctgc g 31 165 43DNA Artificial Sequence Cloning primer 165 ccgcggtcga catgaaaataaaaacaggtg cacgcatcct cgc 43 166 66 DNA Artificial Sequence Cloningprimer 166 gccgtgtcgg attccgaggt gcggccttcg atacgcacgg cataccaagaaagcgggatg 60 ttcggc 66 167 24 DNA Artificial Sequence Cloning primer167 cctcggaatc cgacacggca gggc 24 168 26 DNA Artificial Sequence Cloningprimer 168 gtacagggtc tcccgggtgg cgctgg 26 169 43 DNA ArtificialSequence Cloning primer 169 ccgcgatcga aggccgcacc tcggaatccg acacggcagggcc 43 170 73 DNA Artificial Sequence Cloning primer 170 ggcgcggtacctttgtcatc gtcatcttta taatctgcgg ccgcgtacag ggtctcccgg 60 gtggcgctgg tgg73 171 51 DNA Artificial Sequence Cloning primer 171 gcggcggtaccttattattt gtcatcgtca tctttataat ctgcggccgc g 51 172 79 DNA ArtificialSequence Cloning primer 172 ccgcattaac gacgatgatg ttttccgcct cggctctcgccaaaatcatc gaaggccgca 60 cctcggaatc cgacacggc 79 173 82 DNA ArtificialSequence Cloning primer 173 ccgcggtcga catgaaaata aaaacaggtg cacgcatcctcgcattatcc gcattaacga 60 cgatgatgtt ttccgcctcg gc 82 174 33 DNAArtificial Sequence Cloning primer 174 ccgcgagcga taaaattatt cacctgactgacg 33 175 39 DNA Artificial Sequence Cloning primer 175 gcccgccaggttagcgtcga ggaactcttt caactgacc 39 176 37 DNA Artificial SequenceCloning primer 176 gcggccgcaa gcgataaaat tattcacctg actgacg 37 177 38DNA Artificial Sequence Cloning primer 177 ggcgctgcgg ccgcatcatcatgatcttta taatcgcc 38 178 2465 DNA Artificial Sequence Fusion protein178 gtcgacatgg ggcaacccgg gaacggcagc gccttcttgc tggcacccaa tggaagccat 60gcgccggacc acgacgtcac gcagcaaagg gacgaggtgt gggtggtggg catgggcatc 120gtcatgtctc tcatcgtcct ggccatcgtg tttggcaatg tgctggtcat cacagccatt 180gccaagttcg agcgtctgca gacggtcacc aactacttca tcacttcact ggcctgtgct 240gatctggtca tgggcctagc agtggtgccc tttggggccg cccatattct tatgaaaatg 300tggacttttg gcaacttctg gtgcgagttt tggacttcca ttgatgtgct gtgcgtcacg 360gccagcattg agaccctgtg cgtgatcgca gtggatcgct actttgccat tacttcacct 420ttcaagtacc agagcctgct gaccaagaat aaggcccggg tgatcattct gatggtgtgg 480attgtgtcag gccttayctc cttcttgccc attcagatgc actggtacag ggccacccac 540caggaagcca tcaactgcta tgccaatgag acctgctgtg acttcttcac gaaccaagcc 600tatgccattg cctcttccat cgtgtccttc tacgttcccc tggtgatcat ggtcttcgtc 660tactccaggg tctttcagga ggccaaaagg cagctccaga agattgacaa atctgagggc 720cgcttccatg tccagaacct tagccaggtg gagcaggatg ggcggacggg gcatggactc 780cgcagatctt ccaagttctg cttgaaggag cacaaagccc tcaagacgtt aggcatcatc 840atgggcactt tcaccctctg ctggctgccc ttcttcatcg ttaacattgt gcatgtgatc 900caggataacc tcatccgtaa ggaagtttac atcctcctaa attggatagg ctatgtcaat 960tctggtttca atccccttat ctactgccgg agcccagatt tcaggattgc cttccaggag 1020cttctgtgcc tgcgcaggtc ttctttgaag gcctatggca atggctactc cagcaacggc 1080aacacagggg agcagagtgg atatcacgtg gaacaggaga aagaaaataa actgctgtgt 1140gaagacctcc caggcacgga agactttgtg ggccatcaag gtactgtgcc tagcgataac 1200attgattcac aagggaggaa ttgtagtaca aatgactcac tgctagagcg tggccagacg 1260gtcaccaacc tgcagctcga gggctgcctc gggaacagta agaccgagga ccagcgcaac 1320gaggagaagg cgcagcgtga ggccaacaaa aagatcgaga agcagctgca gaaggacaag 1380caggtctacc gggccacgca ccgcctgctg ctgctgggtg ctggagaatc tggtaaaagc 1440accattgtga agcagatgag gatcctgcat gttaatgggt ttaatggaga cagtgagaag 1500gcaaccaaag tgcaggacat caaaaacaac ctgaaagagg cgattgaaac cattgtggcc 1560gccatgagca acctggtgcc ccccgtggag ctggccaacc ccgagaacca gttcagagtg 1620gactacatcc tgagtgtgat gaacgtgcct gactttgact tccctcccga attctatgag 1680catgccaagg ctctgtggga ggatgaagga gtgcgtgcct gctacgaacg ctccaacgag 1740taccagctga ttgactgtgc ccagtacttc ctggacaaga tcgacgtgat caagcaggct 1800gactatgtgc cgagcgatca ggacctgctt cgctgccgtg tcctgacttc tggaatcttt 1860gagaccaagt tccaggtgga caaagtcaac ttccacatgt ttgacgtggg tggccagcgc 1920gatgaacgcc gcaagtggat ccagtgcttc aacgatgtga ctgccatcat cttcgtggtg 1980gccagcagca gctacaacat ggtcatccgg gaggacaacc agaccaaccg cctgcaggag 2040gctctgaacc tcttcaagag catctggaac aacagatggc tgcgcaccat ctctgtgatc 2100ctgttcctca acaagcaaga tctgctcgct gagaaagtcc ttgctgggaa atcgaagatt 2160gaggactact ttccagaatt tgctcgctac actactcctg aggatgctac tcccgagccc 2220ggagaggacc cacgcgtgac ccgggccaag tacttcattc gagatgagtt tctgaggatc 2280agcactgcca gtggagatgg gcgtcactac tgctaccctc atttcacctg cgctgtggac 2340actgagaaca tccgccgtgt gttcaacgac tgccgtgaca tcattcagcg catgcacctt 2400cgtcagtacg agctgctcat cgattaataa tctagaggat ccccgcgccc tcatccgaaa 2460gggcg 2465 179 2485 DNA Artificial Sequence Transcriptional fusionconstruct 179 gtcgacatgg ggcaacccgg gaacggcagc gccttcttgc tggcacccaatggaagccat 60 gcgccggacc acgacgtcac gcagcaaagg gacgaggtgt gggtggtgggcatgggcatc 120 gtcatgtctc tcatcgtcct ggccatcgtg tttggcaatg tgctggtcatcacagccatt 180 gccaagttcg agcgtctgca gacggtcacc aactacttca tcacttcactggcctgtgct 240 gatctggtca tgggcctagc agtggtgccc tttggggccg cccatattcttatgaaaatg 300 tggacttttg gcaacttctg gtgcgagttt tggacttcca ttgatgtgctgtgcgtcacg 360 gccagcattg agaccctgtg cgtgatcgca gtggatcgct actttgccattacttcacct 420 ttcaagtacc agagcctgct gaccaagaat aaggcccggg tgatcattctgatggtgtgg 480 attgtgtcag gccttayctc cttcttgccc attcagatgc actggtacagggccacccac 540 caggaagcca tcaactgcta tgccaatgag acctgctgtg acttcttcacgaaccaagcc 600 tatgccattg cctcttccat cgtgtccttc tacgttcccc tggtgatcatggtcttcgtc 660 tactccaggg tctttcagga ggccaaaagg cagctccaga agattgacaaatctgagggc 720 cgcttccatg tccagaacct tagccaggtg gagcaggatg ggcggacggggcatggactc 780 cgcagatctt ccaagttctg cttgaaggag cacaaagccc tcaagacgttaggcatcatc 840 atgggcactt tcaccctctg ctggctgccc ttcttcatcg ttaacattgtgcatgtgatc 900 caggataacc tcatccgtaa ggaagtttac atcctcctaa attggataggctatgtcaat 960 tctggtttca atccccttat ctactgccgg agcccagatt tcaggattgccttccaggag 1020 cttctgtgcc tgcgcaggtc ttctttgaag gcctatggca atggctactccagcaacggc 1080 aacacagggg agcagagtgg atatcacgtg gaacaggaga aagaaaataaactgctgtgt 1140 gaagacctcc caggcacgga agactttgtg ggccatcaag gtactgtgcctagcgataac 1200 attgattcac aagggaggaa ttgtagtaca aatgactcac tgctagagcgtggccagacg 1260 gtcaccaacc tgcagtaata atcaaggagg ccctcgagat gggctgcctcgggaacagta 1320 agaccgagga ccagcgcaac gaggagaagg cgcagcgtga ggccaacaaaaagatcgaga 1380 agcagctgca gaaggacaag caggtctacc gggccacgca ccgcctgctgctgctgggtg 1440 ctggagaatc tggtaaaagc accattgtga agcagatgag gatcctgcatgttaatgggt 1500 ttaatggaga cagtgagaag gcaaccaaag tgcaggacat caaaaacaacctgaaagagg 1560 cgattgaaac cattgtggcc gccatgagca acctggtgcc ccccgtggagctggccaacc 1620 ccgagaacca gttcagagtg gactacatcc tgagtgtgat gaacgtgcctgactttgact 1680 tccctcccga attctatgag catgccaagg ctctgtggga ggatgaaggagtgcgtgcct 1740 gctacgaacg ctccaacgag taccagctga ttgactgtgc ccagtacttcctggacaaga 1800 tcgacgtgat caagcaggct gactatgtgc cgagcgatca ggacctgcttcgctgccgtg 1860 tcctgacttc tggaatcttt gagaccaagt tccaggtgga caaagtcaacttccacatgt 1920 ttgacgtggg tggccagcgc gatgaacgcc gcaagtggat ccagtgcttcaacgatgtga 1980 ctgccatcat cttcgtggtg gccagcagca gctacaacat ggtcatccgggaggacaacc 2040 agaccaaccg cctgcaggag gctctgaacc tcttcaagag catctggaacaacagatggc 2100 tgcgcaccat ctctgtgatc ctgttcctca acaagcaaga tctgctcgctgagaaagtcc 2160 ttgctgggaa atcgaagatt gaggactact ttccagaatt tgctcgctacactactcctg 2220 aggatgctac tcccgagccc ggagaggacc cacgcgtgac ccgggccaagtacttcattc 2280 gagatgagtt tctgaggatc agcactgcca gtggagatgg gcgtcactactgctaccctc 2340 atttcacctg cgctgtggac actgagaaca tccgccgtgt gttcaacgactgccgtgaca 2400 tcattcagcg catgcacctt cgtcagtacg agctgctcat cgattaataatctagaggat 2460 ccccgcgccc tcatccgaaa gggcg 2485 180 1146 DNA Homosapien 180 ctcgagatgg gctgcctcgg gaacagtaag accgaggacc agcgcaacgaggagaaggcg 60 cagcgtgagg ccaacaaaaa gatcgagaag cagctgcaga aggacaagcaggtctaccgg 120 gccacgcacc gcctgctgct gctgggtgct ggagaatctg gtaaaagcaccattgtgaag 180 cagatgagga tcctgcatgt taatgggttt aatggagaca gtgagaaggcaaccaaagtg 240 caggacatca aaaacaacct gaaagaggcg attgaaacca ttgtggccgccatgagcaac 300 ctggtgcccc ccgtggagct ggccaacccc gagaaccagt tcagagtggactacatcctg 360 agtgtgatga acgtgcctga ctttgacttc cctcccgaat tctatgagcatgccaaggct 420 ctgtgggagg atgaaggagt gcgtgcctgc tacgaacgct ccaacgagtaccagctgatt 480 gactgtgccc agtacttcct ggacaagatc gacgtgatca agcaggctgactatgtgccg 540 agcgatcagg acctgcttcg ctgccgtgtc ctgacttctg gaatctttgagaccaagttc 600 caggtggaca aagtcaactt ccacatgttt gacgtgggtg gccagcgcgatgaacgccgc 660 aagtggatcc agtgcttcaa cgatgtgact gccatcatct tcgtggtggccagcagcagc 720 tacaacatgg tcatccggga ggacaaccag accaaccgcc tgcaggaggctctgaacctc 780 ttcaagagca tctggaacaa cagatggctg cgcaccatct ctgtgatcctgttcctcaac 840 aagcaagatc tgctcgctga gaaagtcctt gctgggaaat cgaagattgaggactacttt 900 ccagaatttg ctcgctacac tactcctgag gatgctactc ccgagcccggagaggaccca 960 cgcgtgaccc gggccaagta cttcattcga gatgagtttc tgaggatcagcactgccagt 1020 ggagatgggc gtcactactg ctaccctcat ttcacctgcg ctgtggacactgagaacatc 1080 cgccgtgtgt tcaacgactg ccgtgacatc attcagcgca tgcaccttcgtcagtacgag 1140 ctgctc 1146 181 1194 DNA Homo sapien 181 ctcgagatgggctgcctcgg gaacagtaag accgaggacc agcgcaacga ggagaaggcg 60 cagcgtgaggccaacaaaaa gatcgagaag cagctgcaga aggacaagca ggtctaccgg 120 gccacgcaccgcctgctgct gctgggtgct ggagaatctg gtaaaagcac cattgtgaag 180 cagatgaggatcctgcatgt taatgggttt aatggagagg gcggcgaaga ggacccgcag 240 gctgcaaggagcaacagcga tggtgagaag gcaaccaaag tgcaggacat caaaaacaac 300 ctgaaagaggcgattgaaac cattgtggcc gccatgagca acctggtgcc ccccgtggag 360 ctggccaaccccgagaacca gttcagagtg gactacatcc tgagtgtgat gaacgtgcct 420 gactttgacttccctcccga attctatgag catgccaagg ctctgtggga ggatgaagga 480 gtgcgtgcctgctacgaacg ctccaacgag taccagctga ttgactgtgc ccagtacttc 540 ctggacaagatcgacgtgat caagcaggct gactatgtgc cgagcgatca ggacctgctt 600 cgctgccgtgtcctgacttc tggaatcttt gagaccaagt tccaggtgga caaagtcaac 660 ttccacatgtttgacgtggg tggccagcgc gatgaacgcc gcaagtggat ccagtgcttc 720 aacgatgtgactgccatcat cttcgtggtg gccagcagca gctacaacat ggtcatccgg 780 gaggacaaccagaccaaccg cctgcaggag gctctgaacc tcttcaagag catctggaac 840 aacagatggctgcgcaccat ctctgtgatc ctgttcctca acaagcaaga tctgctcgct 900 gagaaagtccttgctgggaa atcgaagatt gaggactact ttccagaatt tgctcgctac 960 actactcctgaggatgctac tcccgagccc ggagaggacc cacgcgtgac ccgggccaag 1020 tacttcattcgagatgagtt tctgaggatc agcactgcca gtggagatgg gcgtcactac 1080 tgctaccctcatttcacctg cgctgtggac actgagaaca tccgccgtgt gttcaacgac 1140 tgccgtgacatcattcagcg catgcacctt cgtcagtacg agctgctcat cgat 1194 182 1089 DNA Homosapien 182 ctcgagatga ctctggagtc catcatggcg tgctgcctga gcgaggaggccaaggaagcc 60 cggcggatca acgacgagat cgagcggcag ctccgcaggg acaagcgggacgcccgccgg 120 gagctcaagc tgctgctgct cgggacagga gagagtggca agagtacgtttatcaagcag 180 atgagaatca tccatgggtc aggatactct gatgaagata aaaggggcttcaccaagctg 240 gtgtatcaga acatcttcac ggccatgcag gccatgatca gagccatggacacactcaag 300 atcccataca agtatgagca caataaggct catgcacaat tagttcgagaagttgatgtg 360 gagaaggtgt ctgcttttga gaatccatat gtagatgcaa taaagagtttatggaatgat 420 cctggaatcc aggaatgcta tgatagacga cgagaatatc aattatctgactctaccaaa 480 tactatctta atgacttgga ccgcgtagct gaccctgcct acctgcctacgcaacaagat 540 gtgcttagag ttcgagtccc caccacaggg atcatcgaat acccctttgacttacaaagt 600 gtcattttca gaatggtcga tgtagggggc caaaggtcag agagaagaaaatggatacac 660 tgctttgaaa atgtcacctc tatcatgttt ctagtagcgc ttagtgaatatgatcaagtt 720 ctcgtggagt cagacaatga gaaccgaatg gaggaaagca aggctctctttagaacaatt 780 atcacatacc cctggttcca gaactcctcg gttattctgt tcttaaacaagaaagatctt 840 ctagaggaga aaatcatgta ttcccatcta gtcgactact tcccagaatatgatggaccc 900 cagagagatg cccaggcagc ccgagaattc attctgaaga tgttcgtggacctgaaccca 960 gacagtgaca aaattatcta ctcccacttc acgtgcgcca cagacaccgagaatatccgc 1020 tttgtctttg ctgccgtcaa ggacaccatc ctccagttga acctgaaggagtacaatctg 1080 gtcatcgat 1089 183 1077 DNA Homo sapien 183 ctcgagatgggctgcaccgt gagcgccgag gacaaggcgg cggccgagcg ctctaagatg 60 atcgacaagaacctgcggga ggacggagag aaggcggcgc gggaggtgaa gttgctgctg 120 ttgggtgctggggagtcagg gaagagcacc atcgtcaagc agatgaagat catccacgag 180 gatggctactccgaggagga atgccggcag taccgggcgg ttgtctacag caacaccatc 240 cagtccatcatggccattgt caaagccatg ggaaacctgc agatcgactt tgccgacccc 300 tccagagcggacgacgccag gcagctattt gcactgtcct gcaccgccga ggagcaaggc 360 gtgctccctgatgacctgtc cggcgtcatc cggaggctct gggctgacca tggtgtgcag 420 gcctgctttggccgctcaag ggaataccag ctcaacgact cagctgccta ctacctgaac 480 gacctggagcgtattgcaca gagtgactac atccccacac agcaagatgt gctacggacc 540 cgcgtaaagaccacggggat cgtggagaca cacttcacct tcaaggacct acacttcaag 600 atgtttgatgtgggtggtca gcggtctgag cggaagaagt ggatccactg ctttgagggc 660 gtcacagccatcatcttctg cgtagccttg agcgcctatg acttggtgct agctgaggac 720 gaggagatgaaccgcatgca tgagagcatg aagctattcg atagcatctg caacaacaag 780 tggttcacagacacgtccat catcctcttc ctcaacaaga aggacctgtt tgaggagaag 840 atcacacacagtcccctgac catctgcttc cctgagtaca caggggccaa caaatatgat 900 gaggcagccagctacatcca gagtaagttt gaggacctga ataagcgcaa agacaccaag 960 gagatctacacgcacttcac gtgcgccacc gacaccaaga acgtgcagtt cgtgtttgac 1020 gccgtcaccgatgtcatcat caagaacaac ctgaaggact gcggcctctt catgcat 1077 184 1155 DNAHomo sapien 184 ctcgagatgt ccggggtggt gcggaccctc agccgctgcc tgctgccggccgaggccggc 60 ggggcccgcg agcgcagggc gggcagcggc gcgcgcgacg cggagcgcgaggcccggagg 120 cgtagccgcg acatcgacgc gctgctggcc cgcgagcggc gcgcggtccggcgcctggtg 180 aagatcctgc tgctgggcgc gggcgagagc ggcaagtcca cgttcctcaagcagatgcgc 240 atcatccacg gccgcgagtt cgaccagaag gcgctgctgg agttccgcgacaccatcttc 300 gacaacatcc tcaagggctc aagggttctt gttgatgcac gagataagcttggcattcct 360 tggcagtatt ctgaaaatga gaagcatggg atgttcctga tggccttcgagaacaaggcg 420 gggctgcctg tggagccggc caccttccag ctgtacgtcc cggccctgagcgcactctgg 480 agggattctg gcatcaggga ggctttcagc cggagaagcg agtttcagctgggggagtcg 540 gtgaagtact tcctggacaa cttggaccgg atcggccagc tgaattactttcctagtaag 600 caagatatcc tgctggctag gaaagccacc aagggaattg tggagcatgacttcgttatt 660 aagaagatcc cctttaagat ggtggatgtg ggcggccagc ggtcccagcgccagaagtgg 720 ttccagtgct tcgacgggat cacgtccatc ctgttcatgg tctcctccagcgagtacgac 780 caggtcctca tggaggacag gcgcaccaac cggctggtgg agtccatgaacatcttcgag 840 accatcgtca acaacaagct cttcttcaac gtctccatca ttctcttcctcaacaagatg 900 gacctcctgg tggagaaggt gaagaccgtg agcatcaaga agcacttcccggacttcagg 960 ggcgacccgc accagctgga ggacgtccag cgctacctgg tccagtgcttcgacaggaag 1020 agacggaacc gcagcaagcc actcttccac cacttcacca ccgccatcgacaccgagaac 1080 gtccgcttcg tgttccatgc tgtgaaagac accatcctgc aggagaacctgaaggacatc 1140 atgctgcaga tcgat 1155 185 3307 DNA Artificial SequenceFusion protein 185 gtcgacatgg ggcaacccgg gaacggcagc gccttcttgctggcacccaa tggaagccat 60 gcgccggacc acgacgtcac gcagcaaagg gacgaggtgtgggtggtggg catgggcatc 120 gtcatgtctc tcatcgtcct ggccatcgtg tttggcaatgtgctggtcat cacagccatt 180 gccaagttcg agcgtctgca gacggtcacc aactacttcatcacttcact ggcctgtgct 240 gatctggtca tgggcctagc agtggtgccc tttggggccgcccatattct tatgaaaatg 300 tggacttttg gcaacttctg gtgcgagttt tggacttccattgatgtgct gtgcgtcacg 360 gccagcattg agaccctgtg cgtgatcgca gtggatcgctactttgccat tacttcacct 420 ttcaagtacc agagcctgct gaccaagaat aaggcccgggtgatcattct gatggtgtgg 480 attgtgtcag gccttayctc cttcttgccc attcagatgcactggtacag ggccacccac 540 caggaagcca tcaactgcta tgccaatgag acctgctgtgacttcttcac gaaccaagcc 600 tatgccattg cctcttccat cgtgtccttc tacgttcccctggtgatcat ggtcttcgtc 660 tactccaggg tctttcagga ggccaaaagg cagctccagaagattgacaa atctgagggc 720 cgcttccatg tccagaacct tagccaggtg gagcaggatgggcggacggg gcatggactc 780 cgcagatctt ccaagttctg cttgaaggag cacaaagccctcaagacgtt aggcatcatc 840 atgggcactt tcaccctctg ctggctgccc ttcttcatcgttaacattgt gcatgtgatc 900 caggataacc tcatccgtaa ggaagtttac atcctcctaaattggatagg ctatgtcaat 960 tctggtttca atccccttat ctactgccgg agcccagatttcaggattgc cttccaggag 1020 cttctgtgcc tgcgcaggtc ttctttgaag gcctatggcaatggctactc cagcaacggc 1080 aacacagggg agcagagtgg atatcacgtg gaacaggagaaagaaaataa actgctgtgt 1140 gaagacctcc caggcacgga agactttgtg ggccatcaaggtactgtgcc tagcgataac 1200 attgattcac aagggaggaa ttgtagtaca aatgactcactgctagagcg tggccagacg 1260 gtcaccaacc tgcagggaca caactcaaaa gagatatcgatgagtcatat tggtactaaa 1320 ttcattcttg ctgaaaaatt taccttcgat cccctaagcaatactctgat tgacaaagaa 1380 gatagtgaag agatcattcg attaggcagc aacgaaagccgaattctttg gctgctggcc 1440 caacgtccaa acgaggtaat ttctcgcaat gatttgcatgactttgtttg gcgagagcaa 1500 ggttttgaag tcgatgattc cagcttaacc caagccatttcgactctgcg caaaatgctc 1560 aaagattcga caaagtcccc acaatacgtc aaaacggttccgaagcgcgg ttaccaattg 1620 atcgcccgag tggaaacggt tgaagaagag atggctcgcgaaaacgaagc tgctcatgac 1680 atctcttaat aatcaaggag gccctcgaga tgggctgcctcgggaacagt aagaccgagg 1740 accagcgcaa cgaggagaag gcgcagcgtg aggccaacaaaaagatcgag aagcagctgc 1800 agaaggacaa gcaggtctac cgggccacgc accgcctgctgctgctgggt gctggagaat 1860 ctggtaaaag caccattgtg aagcagatga ggatcctgcatgttaatggg tttaatggag 1920 acagtgagaa ggcaaccaaa gtgcaggaca tcaaaaacaacctgaaagag gcgattgaaa 1980 ccattgtggc cgccatgagc aacctggtgc cccccgtggagctggccaac cccgagaacc 2040 agttcagagt ggactacatc ctgagtgtga tgaacgtgcctgactttgac ttccctcccg 2100 aattctatga gcatgccaag gctctgtggg aggatgaaggagtgcgtgcc tgctacgaac 2160 gctccaacga gtaccagctg attgactgtg cccagtacttcctggacaag atcgacgtga 2220 tcaagcaggc tgactatgtg ccgagcgatc aggacctgcttcgctgccgt gtcctgactt 2280 ctggaatctt tgagaccaag ttccaggtgg acaaagtcaacttccacatg tttgacgtgg 2340 gtggccagcg cgatgaacgc cgcaagtgga tccagtgcttcaacgatgtg actgccatca 2400 tcttcgtggt ggccagcagc agctacaaca tggtcatccgggaggacaac cagaccaacc 2460 gcctgcagga ggctctgaac ctcttcaaga gcatctggaacaacagatgg ctgcgcacca 2520 tctctgtgat cctgttcctc aacaagcaag atctgctcgctgagaaagtc cttgctggga 2580 aatcgaagat tgaggactac tttccagaat ttgctcgctacactactcct gaggatgcta 2640 ctcccgagcc cggagaggac ccacgcgtga cccgggccaagtacttcatt cgagatgagt 2700 ttctgaggat cagcactgcc agtggagatg ggcgtcactactgctaccct catttcacct 2760 gcgctgtgga cactgagaac atccgccgtg tgttcaacgactgccgtgac atcattcagc 2820 gcatgcacct tcgtcagtac gagctgctca tcgatggacacaactcaaaa gagatatcga 2880 tgagtcatat tggtactaaa ttcattcttg ctgaaaaatttaccttcgat cccctaagca 2940 atactctgat tgacaaagaa gatagtgaag agatcattcgattaggcagc aacgaaagcc 3000 gaattctttg gctgctggcc caacgtccaa acgaggtaatttctcgcaat gatttgcatg 3060 actttgtttg gcgagagcaa ggttttgaag tcgatgattccagcttaacc caagccattt 3120 cgactctgcg caaaatgctc aaagattcga caaagtccccacaatacgtc aaaacggttc 3180 cgaagcgcgg ttaccaattg atcgcccgag tggaaacggttgaagaagag atggctcgcg 3240 aaaacgaagc tgctcatgac atctcttaat aatctagaggatccccgcgc cctcatccga 3300 aagggcg 3307 186 3284 DNA Artificial SequenceFusion protein 186 tctagaggct gtgggtagaa gtgaaacggg gtttaccgataaaaacagaa aatgataaaa 60 aaggactaaa tagtatattt tgatttttga tttttgatttcaaataatac aaatttattt 120 acttatttaa ttgttttgat caattatttt tctgttaaacaaagggagca ttatatggta 180 aagaccatga ttacggattc actggccgtc gttttacaacgtcgtgactg ggaaaaccct 240 ggcgttaccc aacttaatcg ccttgcagca catccccctttcgccagctg gcgtaatagc 300 gaagaggccc gcaccgatcg cccttcccaa cagttgcgcagcctgaatgg cgaatggcgc 360 tttgcctggt ttccggcacc agaagcggtg ccggaaagctggctggagtg cgatcttcct 420 gaggccgata ctgtcgtcgt cccctcaaac tggcagatgcacggttacga tgcgcccatc 480 tacaccaacg tgacctatcc cattacggtc aatccgccgtttgttcccac ggagaatccg 540 acgggttgtt actcgctcac atttaatgtt gatgaaagctggctacagga aggccagacg 600 cgaattattt ttgatggcgt taactcggcg tttcatctgtggtgcaacgg gcgctgggtc 660 ggttacggcc aggacagtcg tttgccgtct gaatttgacctgagcgcatt tttacgcgcc 720 ggagaaaacc gcctcgcggt gatggtgctg cgctggagtgacggcagtta tctggaagat 780 caggatatgt ggcggatgag cggcattttc cgtgacgtctcgttgctgca taaaccgact 840 acacaaatca gcgatttcca tgttgccact cgctttaatgatgatttcag ccgcgctgta 900 ctggaggctg aagttcagat gtgcggcgag ttgcgtgactacctacgggt aacagtttct 960 ttatggcagg gtgaaacgca ggtcgccagc ggcaccgcgcctttcggcgg tgaaattatc 1020 gatgagcgtg gtggttatgc cgatcgcgtc acactacgtctgaacgtcga aaacccgaaa 1080 ctgtggagcg ccgaaatccc gaatctctat cgtgcggtggttgaactgca caccgccgac 1140 ggcacgctga ttgaagcaga agcctgcgat gtcggtttccgcgaggtgcg gattgaaaat 1200 ggtctgctgc tgctgaacgg caagccgttg ctgattcgaggcgttaaccg tcacgagcat 1260 catcctctgc atggtcaggt catggatgag cagacgatggtgcaggatat cctgctgatg 1320 aagcagaaca actttaacgc cgtgcgctgt tcgcattatccgaaccatcc gctgtggtac 1380 acgctgtgcg accgctacgg cctgtatgtg gtggatgaagccaatattga aacccacggc 1440 atggtgccaa tgaatcgtct gaccgatgat ccgcgctggctaccggcgat gagcgaacgc 1500 gtaacgcgaa tggtgcagcg cgatcgtaat cacccgagtgtgatcatctg gtcgctgggg 1560 aatgaatcag gccacggcgc taatcacgac gcgctgtatcgctggatcaa atctgtcgat 1620 ccttcccgcc cggtgcagta tgaaggcggc ggagccgacaccacggccac cgatattatt 1680 tgcccgatgt acgcgcgcgt ggatgaagac cagcccttcccggctgtgcc gaaatggtcc 1740 atcaaaaaat ggctttcgct acctggagag acgcgcccgctgatcctttg cgaatacgcc 1800 cacgcgatgg gtaacagtct tggcggtttc gctaaatactggcaggcgtt tcgtcagtat 1860 ccccgtttac agggcggctt cgtctgggac tgggtggatcagtcgctgat taaatatgat 1920 gaaaacggca acccgtggtc ggcttacggc ggtgattttggcgatacgcc gaacgatcgc 1980 cagttctgta tgaacggtct ggtctttgcc gaccgcacgccgcatccagc gctgacggaa 2040 gcaaaacacc agcagcagtt tttccagttc cgtttatccgggcaaaccat cgaagtgacc 2100 agcgaatacc tgttccgtca tagcgataac gagctcctgcactggatggt ggcgctggat 2160 ggtaagccgc tggcaagcgg tgaagtgcct ctggatgtcgctccacaagg taaacagttg 2220 attgaactgc ctgaactacc gcagccggag agcgccgggcaactctggct cacagtacgc 2280 gtagtgcaac cgaacgcgac cgcatggtca gaagccgggcacatcagcgc ctggcagcag 2340 tggcgtctgg cggaaaacct cagtgtgacg ctccccgccgcgtcccacgc catcccgcat 2400 ctgaccacca gcgaaatgga tttttgcatc gagctgggtaataagcgttg gcaatttaac 2460 cgccagtcag gctttctttc acagatgtgg attggcgataaaaaacaact gctgacgccg 2520 ctgcgcgatc agttcacccg tgcaccgctg gataacgacattggcgtaag tgaagcgacc 2580 cgcattgacc ctaacgcctg ggtcgaacgc tggaaggcggcgggccatta ccaggccgaa 2640 gcagcgttgt tgcagtgcac ggcagataca cttgctgatgcggtgctgat tacgaccgct 2700 cacgcgtggc agcatcaggg gaaaacctta tttatcagccggaaaaccta ccggattgat 2760 ggtagtggtc aaatggcgat taccgttgat gttgaagtggcgagcgatac accgcatccg 2820 gcgcggattg gcctgaactg ccagctggcg caggtagcagagcgggtaaa ctggctcgga 2880 ttagggccgc aagaaaacta tcccgaccgc cttactgccgcctgttttga ccgctgggat 2940 ctgccattgt cagacatgta taccccgtac gtcttcccgagcgaaaacgg tctgcgctgc 3000 gggacgcgcg aattgaatta tggcccacac cagtggcgcggcgacttcca gttcaacatc 3060 agccgctaca gtcaacagca actgatggaa accagccatcgccatctgct gcacgcggaa 3120 gaaggcacat ggctgaatat cgacggtttc catatggggattggtggcga cgactcctgg 3180 agcccgtcag tatcggcgga attccagctg agcgccggtcgctaccatta ccagttggtc 3240 tggtgtcaaa aataataacg ccctcatccg aaagggcgtctaga 3284 187 25 DNA Artificial Sequence Cloning primer 187 ggggcaacccgggaacggca gcgcc 25 188 30 DNA Artificial Sequence Cloning primer 188gcagtgagtc atttgtacta caattcctcc 30 189 37 DNA Artificial SequenceCloning primer 189 cgcggtcgac atggggcaac ccgggaacgg cagcgcc 37 190 67DNA Artificial Sequence Cloning primer 190 ggctcgagct gcaggttggtgaccgtctgg ccacgctcta gcagtgagtc atttgtacta 60 caattcc 67 191 29 DNAArtificial Sequence Cloning primer 191 ggctgcctc gggaacagta agaccgagg 29192 29 DNA Artificial Sequence Cloning primer 192 gagcagctcg tactgacgaaggtgcatgc 29 193 44 DNA Artificial Sequence Cloning primer 193ggaggccctc gagatgggct gcctcgggaa cagtaagacc gagg 44 194 48 DNAArtificial Sequence Cloning primer 194 cctctagatt attatcgatg agcagctcgtactgacgaag gtgcatgc 48 195 37 DNA Artificial Sequence Cloning primer 195ccatcgatga gcagctcgta ctgacgaagg tgcatgc 37 196 26 DNA ArtificialSequence Cloning primer 196 ccggggtggt gcggaccctc agccgc 26 197 28 DNAArtificial Sequence Cloning primer 197 ctgcagcatg atgtccttca ggttctcc 28198 41 DNA Artificial Sequence Cloning primer 198 gcgggctcga gatgtccggggtggtgcgga ccctcagccg c 41 199 39 DNA Artificial Sequence Cloning primer199 gcgccatcga tctgcagcat gatgtccttc aggttctcc 39 200 28 DNA ArtificialSequence Cloning primer 200 gactctggag tccatcatgg cgtgctgc 28 201 29 DNAArtificial Sequence Cloning primer 201 ccagattgta ctccttcagg ttcaactgg29 202 30 DNA Artificial Sequence Cloning primer 202 atgactctggagtccatcat ggcgtgctgc 30 203 42 DNA Artificial Sequence Cloning primer203 gcgccatcga tgaccagatt gtactccttc aggttcaact gg 42 204 29 DNAArtificial Sequence Cloning primer 204 gggctgcacc gtgagcgccg aggacaagg29 205 30 DNA Artificial Sequence Cloning primer 205 ccttcaggttgttcttgatg atgacatcgg 30 206 31 DNA Artificial Sequence Cloning primer206 atgggctgca ccgtgagcgc cgaggacaag g 31 207 55 DNA Artificial SequenceCloning primer 207 gcgccatcga tgaagaggcc gcagtccttc aggttgttcttgatgatgac atcgg 55 208 29 DNA Artificial Sequence Cloning primer 208gggctgcctc gggaacagta agaccgagg 29 209 29 DNA Artificial SequenceCloning primer 209 gagcagctcg tactgacgaa ggtgcatgc 29 210 31 DNAArtificial Sequence Cloning primer 210 atgggctgcc tcgggaacag taagaccgagg 31 211 40 DNA Artificial Sequence Cloning primer 211 gcgccatcgatgagcagctc gtactgacga aggtgcatgc 40 212 107 DNA Artificial SequenceCloning primer 212 ggctcgaggg cctccttgat tattactcga gggcctccttgattattact gcaggttggt 60 gaccgtctgg ccacgctcta gcagtgagtc atttgtactacaattcc 107 213 61 DNA Artificial Sequence Cloning primer 213 ccctgcaggttggtgaccgt ctggccacgc tctagcagtg agtcatttgt actacaattc 60 c 61 214 38DNA Artificial Sequence Cloning primer 214 ggacacaact caaaagagatatcgatgagt catattgg 38 215 29 DNA Artificial Sequence Cloning primer 215gagatgtcat gagcagcttc gttttcgcg 29 216 103 DNA Artificial SequenceCloning primer 216 gcgtggccag acggtcacca acctgcaggg acacaactcaaaagagatat cgcggggatc 60 ctctagatta ttaagagatg tcatgagcag cttcgttttc gcg103 217 32 DNA Artificial Sequence Cloning primer 217 ggctgtgggtagaagtgaaa cggggtttac cg 32 218 32 DNA Artificial Sequence Cloningprimer 218 ctttaccata taatgctccc tttgtttaac ag 32 219 43 DNA ArtificialSequence Cloning primer 219 cgcggtctag aggctgtggg tagaagtgaa acggggtttaccg 43 220 60 DNA Artificial Sequence Cloning primer 220 cgacggccagtgaatccgta atcatggtct ttaccatata atgctccctt tgtttaacag 60 221 27 DNAArtificial Sequence Cloning primer 221 ccatgattac ggattcactg gccgtcg 27222 27 DNA Artificial Sequence Cloning primer 222 ccagaccaac tggtaatggtagcgacc 27 223 35 DNA Artificial Sequence Cloning primer 223 ggtaaagaccatgattacgg attcactggc cgtcg 35 224 77 DNA Artificial Sequence Cloningprimer 224 gcgcctctag aaatacgccc tttcggatga gggcgttatt atttttgacaccagaccaac 60 tggtaatggt agcgacc 77 225 89 DNA Artificial SequenceCloning primer 225 cgcggatgca tatgaaaata aaaacaggtg cacgcatcctcgcattatcc gcattaacga 60 cgatgatgtt ttccgcctcg gctctcgcc 89 226 69 DNAArtificial Sequence Cloning primer 226 cgtcgaccga ggcctgcagg cgggcttcgatgattttggc gagagccgag gcggaaaaca 60 tcatcgtcg 69 227 81 DNA ArtificialSequence Cloning primer 227 cgaagcccgc ctgcaggcct cggtcgacgc cgaatctagagattataaag atgacgatga 60 caaataataa gctagcggcg c 81 228 27 DNAArtificial Sequence Cloning primer 228 gcgccgctag cttattattt gtcatcg 27229 26 DNA Artificial Sequence Cloning primer 229 ggtgcacgca tcctcgcattatccgc 26 230 30 DNA Artificial Sequence Cloning primer 230 ggcgttttccatggtggcgg caatacgtgg 30 231 52 DNA Artificial Sequence Cloning primer231 cgcggatgca tatgaaaata aaaacaggtg cacgcatcct cgcattatcc gc 52 232 82DNA Artificial Sequence Cloning primer 232 ccgaggcctg caggcgggcttcgatacgca cggcatacca gaaagcggac tgggcgtttt 60 ccatggtggc ggcaatacgt gg82 233 87 DNA Artificial Sequence Cloning primer 233 gcgccgctagcttattattt gtcatcgtca tctttataat ctctagattc ggcgtcgacc 60 gaggcctgcaggcgggcttc gatacgc 87 234 26 DNA Artificial Sequence Cloning primer 234cctgactgac gacagttttg acacgg 26 235 32 DNA Artificial Sequence Cloningprimer 235 cctttagaca gtgcacccac tttggttgcc gc 32 236 78 DNA ArtificialSequence Cloning primer 236 cgcggctgca ggcctcggtc gacgccgaat ctagaagcgataaaattatt cacctgactg 60 acgacagttt tgacacgg 78 237 95 DNA ArtificialSequence Cloning primer 237 gcgccgctag cttattattt gtcatcgtca tctttataatccgccaggtt ctctttcaac 60 tgacctttag acagtgcacc cactttggtt gccgc 95 238216 DNA Artificial Sequence Cloning primer 238 gaattcaggc gctttttagactggtcgtaa tgaaattcag gaggttctgc atatgaaaat 60 aaaaacaggt gcacgcatcctcgcattatc cgcattaacg acgatgatgt tttccgcctc 120 ggctctcgcc aaaatcatcgaagcccgcct gcaggcctcg gtcgacgccg aatctagaga 180 ttataaagat gacgatgacaaataataagc tagagg 216 239 202 DNA Artificial Sequence Cloning primer 239ccatacccgt ttttttgggc tagcaggagg ccctgcatat gaaaataaaa acaggtgcac 60gcatcctcgc attatccgca ttaacgacga tgatgttttc cgcctcggct ctcgccaaaa 120tcatcgaagc ccgcctgcag gcctcggtcg acgccgaatc tagagattat aaagatgacg 180atgacaaata ataagctaga gg 202 240 182 DNA Artificial Sequence Cloningprimer 240 aggaggttct gcatatgaaa ataaaaacag gtgcacgcat cctcgcattatccgcattaa 60 cgacgatgat gttttccgcc tcggctctcg ccaaaatcat cgaagcccgcctgcaggcct 120 cggtcgacgc cgaatctaga gattataaag atgacgatga caaataataagctagaggta 180 cc 182 241 182 DNA Artificial Sequence Cloning primer 241aggaggttct gcatatgaaa ataaaaacag gtgcacgcat cctcgcatta tccgcattaa 60cgacgatgat gttttccgcc tcggctctcg ccaaaatcat cgaagcccgc ctgcaggcct 120cggtcgacgc cgaatctaga gattataaag atgacgatga caaataataa gctagaggta 180 cc182 242 1080 DNA Artificial Sequence Expression vector 242 gaattcaggcgctttttaga ctggtcgtaa tgaaattcag gaggttctgc atatgaaaat 60 aaaaacaggtgcacgcatcc tcgcattatc cgcattaacg acgatgatgt tttccgcctc 120 ggctctcgccaaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta 180 taacggtctcgctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt 240 tgagcatccggataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc 300 tgacattatcttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc 360 tgaaatcaccccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt 420 acgttacaacggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta 480 taacaaagatctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa 540 agaactgaaagcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac 600 ctggccgctgattgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga 660 cattaaagacgtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga 720 cctgattaaaaacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt 780 taataaaggcgaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac 840 cagcaaagtgaattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc 900 gttcgttggcgtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcgaa 960 agagttcctcgaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa 1020 accgctgggtgccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat 1080 243 1196 DNAArtificial Sequence Expression vector 243 ccatacccgt ttttttgggctagcaggagg ccctgcatat gaaaataaaa acaggtgcac 60 gcatcctcgc attatccgcattaacgacga tgatgttttc cgcctcggct ctcgccaaaa 120 tcgaagaagg taaactggtaatctggatta acggcgataa aggctataac ggtctcgctg 180 aagtcggtaa gaaattcgagaaagataccg gaattaaagt caccgttgag catccggata 240 aactggaaga gaaattcccacaggttgcgg caactggcga tggccctgac attatcttct 300 gggcacacga ccgctttggtggctacgctc aatctggcct gttggctgaa atcaccccgg 360 acaaagcgtt ccaggacaagctgtatccgt ttacctggga tgccgtacgt tacaacggca 420 agctgattgc ttacccgatcgctgttgaag cgttatcgct gatttataac aaagatctgc 480 tgccgaaccc gccaaaaacctgggaagaga tcccggcgct ggataaagaa ctgaaagcga 540 aaggtaagag cgcgctgatgttcaacctgc aagaaccgta cttcacctgg ccgctgattg 600 ctgctgacgg gggttatgcgttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg 660 gcgtggataa cgctggcgcgaaagcgggtc tgaccttcct ggttgacctg attaaaaaca 720 aacacatgaa tgcagacaccgattactcca tcgcagaagc tgcctttaat aaaggcgaaa 780 cagcgatgac catcaacggcccgtgggcat ggtccaacat cgacaccagc aaagtgaatt 840 atggtgtaac ggtactgccgaccttcaagg gtcaaccatc caaaccgttc gttggcgtgc 900 tgagcgcagg tattaacgccgccagtccga acaaagagct ggcgaaagag ttcctcgaaa 960 actatctgct gactgatgaaggtctggaag cggttaataa agacaaaccg ctgggtgccg 1020 tagcgctgaa gtcttacgaggaagagttgg cgaaagatcc acgtattgcc gccaccatgg 1080 aaaacgccca gtccgctttctggtatgccg tgcgtatcga agcccgcctg caggcctcgg 1140 tcgacgccga atctagagattataaagatg acgatgacaa ataataagct agagga 1196 244 1171 DNA ArtificialSequence Expression vector 244 aggaggttct gcatatgaaa ataaaaacaggtgcacgcat cctcgcatta tccgcattaa 60 cgacgatgat gttttccgcc tcggctctcgccaaaatcga agaaggtaaa ctggtaatct 120 ggattaacgg cgataaaggc tataacggtctcgctgaagt cggtaagaaa ttcgagaaag 180 ataccggaat taaagtcacc gttgagcatccggataaact ggaagagaaa ttcccacagg 240 ttgcggcaac tggcgatggc cctgacattatcttctgggc acacgaccgc tttggtggct 300 acgctcaatc tggcctgttg gctgaaatcaccccggacaa agcgttccag gacaagctgt 360 atccgtttac ctgggatgcc gtacgttacaacggcaagct gattgcttac ccgatcgctg 420 ttgaagcgtt atcgctgatt tataacaaagatctgctgcc gaacccgcca aaaacctggg 480 aagagatccc ggcgctggat aaagaactgaaagcgaaagg taagagcgcg ctgatgttca 540 acctgcaaga accgtacttc acctggccgctgattgctgc tgacgggggt tatgcgttca 600 agtatgaaaa cggcaagtac gacattaaagacgtgggcgt ggataacgct ggcgcgaaag 660 cgggtctgac cttcctggtt gacctgattaaaaacaaaca catgaatgca gacaccgatt 720 actccatcgc agaagctgcc tttaataaaggcgaaacagc gatgaccatc aacggcccgt 780 gggcatggtc caacatcgac accagcaaagtgaattatgg tgtaacggta ctgccgacct 840 tcaagggtca accatccaaa ccgttcgttggcgtgctgag cgcaggtatt aacgccgcca 900 gtccgaacaa agagctggcg aaagagttcctcgaaaacta tctgctgact gatgaaggtc 960 tggaagcggt taataaagac aaaccgctgggtgccgtagc gctgaagtct tacgaggaag 1020 agttggcgaa agatccacgt attgccgccaccatggaaaa cgcccagtcc gctttctggt 1080 atgccgtgcg tatcgaagcc cgcctgcaggcctcggtcga cgccgaatct agagattata 1140 aagatgacga tgacaaataa taagctagag g1171 245 1171 DNA Artificial Sequence Expression vector 245 aggaggttctgcatatgaaa ataaaaacag gtgcacgcat cctcgcatta tccgcattaa 60 cgacgatgatgttttccgcc tcggctctcg ccaaaatcga agaaggtaaa ctggtaatct 120 ggattaacggcgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag 180 ataccggaattaaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg 240 ttgcggcaactggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct 300 acgctcaatctggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt 360 atccgtttacctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg 420 ttgaagcgttatcgctgatt tataacaaag atctgctgcc gaacccgcca aaaacctggg 480 aagagatcccggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca 540 acctgcaagaaccgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca 600 agtatgaaaacggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag 660 cgggtctgaccttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt 720 actccatcgcagaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt 780 gggcatggtccaacatcgac accagcaaag tgaattatgg tgtaacggta ctgccgacct 840 tcaagggtcaaccatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca 900 gtccgaacaaagagctggcg aaagagttcc tcgaaaacta tctgctgact gatgaaggtc 960 tggaagcggttaataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag 1020 agttggcgaaagatccacgt attgccgcca ccatggaaaa cgcccagtcc gctttctggt 1080 atgccgtgcgtatcgaagcc cgcctgcagg cctcggtcga cgccgaatct agagattata 1140 aagatgacgatgacaaataa taagctagag g 1171 246 392 DNA Artificial Sequence Fusionprotein 246 tagcaggagg ccctgcaggc ctcggtcgac gccgaatcta gaagcgataaaattattcac 60 ctgactgacg acagttttga cacggatgta ctcaaagcgg acggggcgatcctcgtcgat 120 ttctgggcag agtggtgcgg tccgtgcaaa atgatcgccc cgattctggatgaaatcgct 180 gacgaatatc agggcaaact gaccgttgca aaactgaaca tcgatcaaaaccctggcact 240 gcgccgaaat atggcatccg tggtatcccg actctgctgc tgttcaaaaacggtgaagtg 300 gcggcaacca aagtgggtgc actgtctaaa ggtcagttga aagagaacctggcggattat 360 aaagatgacg atgacaaata ataagctaga gg 392 247 426 DNAArtificial Sequence Fusion protein 247 gaattcaggc gctttttaga ctggtcgtaatgaaattcag gaggttctgc aggcctcggt 60 cgacgccgaa tctagaagcg ataaaattattcacctgact gacgacagtt ttgacacgga 120 tgtactcaaa gcggacgggg cgatcctcgtcgatttctgg gcagagtggt gcggtccgtg 180 caaaatgatc gccccgattc tggatgaaatcgctgacgaa tatcagggca aactgaccgt 240 tgcaaaactg aacatcgatc aaaaccctggcactgcgccg aaatatggca tccgtggtat 300 cccgactctg ctgctgttca aaaacggtgaagtggcggca accaaagtgg gtgcactgtc 360 taaaggtcag ttgaaagaga acctggcggattataaagat gacgatgaca aataataagc 420 tagagg 426 248 392 DNA ArtificialSequence Fusion protein 248 aggaggttct gcaggcctcg gtcgacgccg aatctagaagcgataaaatt attcacctga 60 ctgacgacag ttttgacacg gatgtactca aagcggacggggcgatcctc gtcgatttct 120 gggcagagtg gtgcggtccg tgcaaaatga tcgccccgattctggatgaa atcgctgacg 180 aatatcaggg caaactgacc gttgcaaaac tgaacatcgatcaaaaccct ggcactgcgc 240 cgaaatatgg catccgtggt atcccgactc tgctgctgttcaaaaacggt gaagtggcgg 300 caaccaaagt gggtgcactg tctaaaggtc agttgaaagagaacctggcg gattataaag 360 atgacgatga caaataataa gctagaggta cc 392 249392 DNA Artificial Sequence Fusion protein 249 aggaggttct gcaggcctcggtcgacgccg aatctagaag cgataaaatt attcacctga 60 ctgacgacag ttttgacacggatgtactca aagcggacgg ggcgatcctc gtcgatttct 120 gggcagagtg gtgcggtccgtgcaaaatga tcgccccgat tctggatgaa atcgctgacg 180 aatatcaggg caaactgaccgttgcaaaac tgaacatcga tcaaaaccct ggcactgcgc 240 cgaaatatgg catccgtggtatcccgactc tgctgctgtt caaaaacggt gaagtggcgg 300 caaccaaagt gggtgcactgtctaaaggtc agttgaaaga gaacctggcg gattataaag 360 atgacgatga caaataataagctagaggta cc 392 250 528 DNA Artificial Sequence Fusion protein 250gaattcaggc gctttttaga ctggtcgtaa tgaaattcag gaggttctgc atatgaaaat 60aaaaacaggt gcacgcatcc tcgcattatc cgcattaacg acgatgatgt tttccgcctc 120ggctctcgcc aaaatcatcg aagcccgcct gcaggcctcg gtcgacgccg aatctagaag 180cgataaaatt attcacctga ctgacgacag ttttgacacg gatgtactca aagcggacgg 240ggcgatcctc gtcgatttct gggcagagtg gtgcggtccg tgcaaaatga tcgccccgat 300tctggatgaa atcgctgacg aatatcaggg caaactgacc gttgcaaaac tgaacatcga 360tcaaaaccct ggcactgcgc cgaaatatgg catccgtggt atcccgactc tgctgctgtt 420caaaaacggt gaagtggcgg caaccaaagt gggtgcactg tctaaaggtc agttgaaaga 480gaacctggcg gattataaag atgacgatga caaataataa gctagagg 528 251 514 DNAArtificial Sequence Fusion protein 251 ccatacccgt ttttttgggc tagcaggaggccctgcatat gaaaataaaa acaggtgcac 60 gcatcctcgc attatccgca ttaacgacgatgatgttttc cgcctcggct ctcgccaaaa 120 tcatcgaagc ccgcctgcag gcctcggtcgacgccgaatc tagaagcgat aaaattattc 180 acctgactga cgacagtttt gacacggatgtactcaaagc ggacggggcg atcctcgtcg 240 atttctgggc agagtggtgc ggtccgtgcaaaatgatcgc cccgattctg gatgaaatcg 300 ctgacgaata tcagggcaaa ctgaccgttgcaaaactgaa catcgatcaa aaccctggca 360 ctgcgccgaa atatggcatc cgtggtatcccgactctgct gctgttcaaa aacggtgaag 420 tggcggcaac caaagtgggt gcactgtctaaaggtcagtt gaaagagaac ctggcggatt 480 ataaagatga cgatgacaaa taataagctagagg 514 252 494 DNA Artificial Sequence Fusion protein 252 aggaggttctgcatatgaaa ataaaaacag gtgcacgcat cctcgcatta tccgcattaa 60 cgacgatgatgttttccgcc tcggctctcg ccaaaatcat cgaagcccgc ctgcaggcct 120 cggtcgacgccgaatctaga agcgataaaa ttattcacct gactgacgac agttttgaca 180 cggatgtactcaaagcggac ggggcgatcc tcgtcgattt ctgggcagag tggtgcggtc 240 cgtgcaaaatgatcgccccg attctggatg aaatcgctga cgaatatcag ggcaaactga 300 ccgttgcaaaactgaacatc gatcaaaacc ctggcactgc gccgaaatat ggcatccgtg 360 gtatcccgactctgctgctg ttcaaaaacg gtgaagtggc ggcaaccaaa gtgggtgcac 420 tgtctaaaggtcagttgaaa gagaacctgg cggattataa agatgacgat gacaaataat 480 aagctagaggtacc 494 253 494 DNA Artificial Sequence Cloning primer 253 aggaggttctgcatatgaaa ataaaaacag gtgcacgcat cctcgcatta tccgcattaa 60 cgacgatgatgttttccgcc tcggctctcg ccaaaatcat cgaagcccgc ctgcaggcct 120 cggtcgacgccgaatctaga agcgataaaa ttattcacct gactgacgac agttttgaca 180 cggatgtactcaaagcggac ggggcgatcc tcgtcgattt ctgggcagag tggtgcggtc 240 cgtgcaaaatgatcgccccg attctggatg aaatcgctga cgaatatcag ggcaaactga 300 ccgttgcaaaactgaacatc gatcaaaacc ctggcactgc gccgaaatat ggcatccgtg 360 gtatcccgactctgctgctg ttcaaaaacg gtgaagtggc ggcaaccaaa gtgggtgcac 420 tgtctaaaggtcagttgaaa gagaacctgg cggattataa agatgacgat gacaaataat 480 aagctagaggtacc 494 254 1521 DNA Artificial Sequence Fusion protein 254 gaattcaggcgctttttaga ctggtcgtaa tgaaattcag gaggttctgc atatgaaaat 60 aaaaacaggtgcacgcatcc tcgcattatc cgcattaacg acgatgatgt tttccgcctc 120 ggctctcgccaaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta 180 taacggtctcgctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt 240 tgagcatccggataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc 300 tgacattatcttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc 360 tgaaatcaccccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt 420 acgttacaacggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta 480 taacaaagatctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa 540 agaactgaaagcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac 600 ctggccgctgattgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga 660 cattaaagacgtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga 720 cctgattaaaaacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt 780 taataaaggcgaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac 840 cagcaaagtgaattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc 900 gttcgttggcgtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcgaa 960 agagttcctcgaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa 1020 accgctgggtgccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat 1080 tgccgccaccatggaaaacg cccagtccgc tttctggtat gccgtgcgta tcgaagcccg 1140 cctgcaggcctcggtcgacg ccgaatctag aagcgataaa attattcacc tgactgacga 1200 cagttttgacacggatgtac tcaaagcgga cggggcgatc ctcgtcgatt tctgggcaga 1260 gtggtgcggtccgtgcaaaa tgatcgcccc gattctggat gaaatcgctg acgaatatca 1320 gggcaaactgaccgttgcaa aactgaacat cgatcaaaac cctggcactg cgccgaaata 1380 tggcatccgtggtatcccga ctctgctgct gttcaaaaac ggtgaagtgg cggcaaccaa 1440 agtgggtgcactgtctaaag gtcagttgaa agagaacctg gcggattata aagatgacga 1500 tgacaaataataagctagag g 1521 255 1500 DNA Artificial Sequence Fusion protein 255ccatacccgt ttttttgggc tagcaggagg ccctgcatat gaaaataaaa acaggtgcac 60gcatcctcgc attatccgca ttaacgacga tgatgttttc cgcctcggct ctcgccaaaa 120tcgaagaagg taaactggta atctggatta acggcgataa aggctataac ggtctcgctg 180aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata 240aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac attatcttct 300gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg 360acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca 420agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc 480tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga 540aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg 600ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg 660gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca 720aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa 780cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt 840atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc 900tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcgaaagag ttcctcgaaa 960actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg 1020tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccaccatgg 1080aaaacgccca gtccgctttc tggtatgccg tgcgtatcga agcccgcctg caggcctcgg 1140tcgacgccga atctagaagc gataaaatta ttcacctgac tgacgacagt tttgacacgg 1200atgtactcaa agcggacggg gcgatcctcg tcgatttctg ggcagagtgg tgcggtccgt 1260gcaaaatgat cgccccgatt ctggatgaaa tcgctgacga atatcagggc aaactgaccg 1320ttgcaaaact gaacatcgat caaaaccctg gcactgcgcc gaaatatggc atccgtggta 1380tcccgactct gctgctgttc aaaaacggtg aagtggcggc aaccaaagtg ggtgcactgt 1440ctaaaggtca gttgaaagag aacctggcgg attataaaga tgacgatgac aaataataag 1500256 1476 DNA Artificial Sequence Fusion protein 256 aggaggttctgcatatgaaa ataaaaacag gtgcacgcat cctcgcatta tccgcattaa 60 cgacgatgatgttttccgcc tcggctctcg ccaaaatcga agaaggtaaa ctggtaatct 120 ggattaacggcgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag 180 ataccggaattaaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg 240 ttgcggcaactggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct 300 acgctcaatctggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt 360 atccgtttacctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg 420 ttgaagcgttatcgctgatt tataacaaag atctgctgcc gaacccgcca aaaacctggg 480 aagagatcccggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca 540 acctgcaagaaccgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca 600 agtatgaaaacggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag 660 cgggtctgaccttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt 720 actccatcgcagaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt 780 gggcatggtccaacatcgac accagcaaag tgaattatgg tgtaacggta ctgccgacct 840 tcaagggtcaaccatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca 900 gtccgaacaaagagctggcg aaagagttcc tcgaaaacta tctgctgact gatgaaggtc 960 tggaagcggttaataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag 1020 agttggcgaaagatccacgt attgccgcca ccatggaaaa cgcccagtcc gctttctggt 1080 atgccgtgcgtatcgaagcc cgcctgcagg cctcggtcga cgccgaatct agaagcgata 1140 aaattattcacctgactgac gacagttttg acacggatgt actcaaagcg gacggggcga 1200 tcctcgtcgatttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc ccgattctgg 1260 atgaaatcgctgacgaatat cagggcaaac tgaccgttgc aaaactgaac atcgatcaaa 1320 accctggcactgcgccgaaa tatggcatcc gtggtatccc gactctgctg ctgttcaaaa 1380 acggtgaagtggcggcaacc aaagtgggtg cactgtctaa aggtcagttg aaagagaacc 1440 tggcggattataaagatgac gatgacaaat aataag 1476 257 1476 DNA Artificial SequenceFusion protein 257 aggaggttct gcatatgaaa ataaaaacag gtgcacgcatcctcgcatta tccgcattaa 60 cgacgatgat gttttccgcc tcggctctcg ccaaaatcgaagaaggtaaa ctggtaatct 120 ggattaacgg cgataaaggc tataacggtc tcgctgaagtcggtaagaaa ttcgagaaag 180 ataccggaat taaagtcacc gttgagcatc cggataaactggaagagaaa ttcccacagg 240 ttgcggcaac tggcgatggc cctgacatta tcttctgggcacacgaccgc tttggtggct 300 acgctcaatc tggcctgttg gctgaaatca ccccggacaaagcgttccag gacaagctgt 360 atccgtttac ctgggatgcc gtacgttaca acggcaagctgattgcttac ccgatcgctg 420 ttgaagcgtt atcgctgatt tataacaaag atctgctgccgaacccgcca aaaacctggg 480 aagagatccc ggcgctggat aaagaactga aagcgaaaggtaagagcgcg ctgatgttca 540 acctgcaaga accgtacttc acctggccgc tgattgctgctgacgggggt tatgcgttca 600 agtatgaaaa cggcaagtac gacattaaag acgtgggcgtggataacgct ggcgcgaaag 660 cgggtctgac cttcctggtt gacctgatta aaaacaaacacatgaatgca gacaccgatt 720 actccatcgc agaagctgcc tttaataaag gcgaaacagcgatgaccatc aacggcccgt 780 gggcatggtc caacatcgac accagcaaag tgaattatggtgtaacggta ctgccgacct 840 tcaagggtca accatccaaa ccgttcgttg gcgtgctgagcgcaggtatt aacgccgcca 900 gtccgaacaa agagctggcg aaagagttcc tcgaaaactatctgctgact gatgaaggtc 960 tggaagcggt taataaagac aaaccgctgg gtgccgtagcgctgaagtct tacgaggaag 1020 agttggcgaa agatccacgt attgccgcca ccatggaaaacgcccagtcc gctttctggt 1080 atgccgtgcg tatcgaagcc cgcctgcagg cctcggtcgacgccgaatct agaagcgata 1140 aaattattca cctgactgac gacagttttg acacggatgtactcaaagcg gacggggcga 1200 tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaaaatgatcgcc ccgattctgg 1260 atgaaatcgc tgacgaatat cagggcaaac tgaccgttgcaaaactgaac atcgatcaaa 1320 accctggcac tgcgccgaaa tatggcatcc gtggtatcccgactctgctg ctgttcaaaa 1380 acggtgaagt ggcggcaacc aaagtgggtg cactgtctaaaggtcagttg aaagagaacc 1440 tggcggatta taaagatgac gatgacaaat aataag 1476

1. A method of identifying an agent that specifically binds a targetcompound, comprising contacting a minicell displaying said targetcompound with a library of compounds, and identifying an agent in saidlibrary that binds said target compound.
 2. The method of claim 1,wherein said minicell is a eubacterial minicell, a poroplast, aspheroplast or a protoplast.
 3. The method of claim 1, wherein saidlibrary of compounds is a protein library.
 4. The method of claim 3,wherein said protein library is selected from the group consisting of aphage display library, a phagemid display library, a baculoviruslibrary, a yeast display library, and a ribosomal display library. 5.The method of claim 1, wherein said library of compounds is selectedfrom the group consisting of a library of aptamers, a library ofsynthetic peptides and a library of small molecules.
 6. The method ofclaim 1, wherein said target compound is a target polypeptide.
 7. Themethod of claim 6, wherein said minicell comprises an expressionconstruct comprising expression sequences operably linked to an ORFencoding said target polypeptide.
 8. The method of claim 6, wherein saidtarget polypeptide is a membrane protein.
 9. The method of claim 8,wherein said membrane protein is a receptor or a channel protein. 10.The method of claim 8, wherein said membrane protein is an enzyme. 11.The method of claim 1, wherein said target compound is a membrane fusionprotein, said membrane fusion protein comprising a first polypeptide,wherein said first polypeptide comprises at least one transmembranedomain or at least one membrane anchoring domain; and a secondpolypeptide, wherein said second polypeptide comprises amino acidsequences derived from a target polypeptide.
 12. The method of claim 1,wherein said method further comprises comparing the activity of saidtarget compound in the presence of said agent to the activity of saidtarget compound in the absence of said agent.
 13. The method of claim12, wherein said activity of said target compound is an enzyme activity.14. The method of claim 13, wherein said enzyme is selected from thegroup consisting of an oxidoreductase, a transferase, a hydrolase, alyase, an isomerase, a ligase and a synthetase.
 15. The method of claim12, wherein said activity of said target compound is a binding activity.16. The method of claim 15, further comprising comparing the binding ofsaid agent to said target compound to the binding of a known ligand ofsaid target compound.
 17. The method of claim 16, wherein a competitionassay is used for said comparing.
 18. A device comprising microchipsoperatively associated with a biosensor comprising a set of minicells ina prearranged pattern, wherein said each of said microchips comprise orcontact a minicell, wherein each of said minicell displays a differenttarget compound, and wherein binding of a ligand to a target compoundresults in an increased or decreased signal.
 19. A method of identifyingan agent that specifically binds a target compound, comprisingcontacting the device of claim 18 with a library of compounds, anddetecting a signal from said device, wherein said signal changes as afunction of the binding of an agent to said target compound.
 20. Amethod of identifying an agent that specifically blocks the binding of atarget compound to its ligand, comprising contacting the device of claim18 with a library of compounds, and detecting a signal from said device,wherein said signal changes as a function of the binding of an agent tosaid target compound.